Photonic structure-based devices and compositions for use in luminescent imaging of multiple sites within a pixel, and methods of using the same

ABSTRACT

A device for luminescent imaging includes an array of imaging pixels, a photonic structure over the array of imaging pixels, and an array of features over the photonic structure. A first feature of the array of features is over a first pixel of the array of imaging pixels, and a second feature of the array of features is over the first pixel and spatially displaced from the first feature. A first luminophore is within or over the first feature, and a second luminophore is within or over the second feature. The device includes a radiation source to generate first photons having a first characteristic at a first time, and generate second photons having a second characteristic at a second time. The first pixel selectively receives luminescence emitted by the first and second luminophores responsive to the first photons at the first time and second photons at the second time, respectively.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 National Stage application ofInternational Patent Application No. PCT/US2017/028883, filed on Apr.21, 2017, which claims the benefit of U.S. Provisional PatentApplication No. 62/326,568, filed Apr. 22, 2016 and entitled “PHOTONICSTRUCTURE-BASED DEVICES AND COMPOSITIONS FOR USE IN LUMINESCENT IMAGINGOF MULTIPLE SITES WITHIN A PIXEL, AND METHODS OF USING THE SAME;” eachof the aforementioned applications is incorporated by reference in itsentirety.

FIELD

This application generally relates to luminescent imaging.

BACKGROUND

Certain state-of-the-art sequencing tools developed by industry leadersrely on various “sequencing by synthesis (SBS)” chemistries to determinea polynucleotide sequence, such as a DNA or RNA sequence. Sequencing caninvolve using luminescent imaging, such as a fluorescent microscopysystem, to identify nucleotides or localized clusters of identicalnucleotides by emission wavelength of their respective fluorescentmarkers. Although some SBS chemistries under development can require asfew as a single dye, multiple fluorescent dyes (up to four) aregenerally used in commercial systems so as to uniquely identify thenucleotides in a polynucleotide, such as A, G, C, and T nucleotides inDNA.

SUMMARY

Embodiments of the present invention provide photonic structure-baseddevices and compositions for use in luminescent imaging of multiplesites within a pixel, and methods of using the same.

Under one aspect, a device is provided for use in luminescent imaging.The device can include an array of imaging pixels, and a photonicstructure disposed over the array of imaging pixels. The device furthercan include an array of features disposed over the photonic structure. Afirst feature of the array of features can be disposed over a firstpixel of the array of imaging pixels, and a second feature of the arrayof features can be disposed over the first pixel and spatially displacedfrom the first feature. A first luminophore can be disposed within orover the first feature, and a second luminophore can be disposed withinor over the second feature. The device further can include a radiationsource configured to generate first photons having a firstcharacteristic at a first time, and configured to generate secondphotons having a second characteristic at a second time. The secondcharacteristic can be different than the first characteristic, and thesecond time can be different than the first time. The first pixel canselectively receive luminescence emitted by the first luminophoreresponsive to the first photons at the first time, and can selectivelyreceive luminescence emitted by the second luminophore responsive to thesecond photons at the second time.

Optionally, the first photons having the first characteristic generate afirst resonant pattern within the photonic structure at the first time,the first resonant pattern selectively exciting the first luminophorerelative to the second luminophore. Optionally, the second photonshaving the second characteristic generate a second resonant patternwithin the photonic structure at the second time, the second resonantpattern selectively exciting the second luminophore relative to thefirst luminophore.

Additionally, or alternatively, the array of imaging pixels, thephotonic structure, and the array of features optionally aremonolithically integrated with one another.

Additionally, or alternatively, the photonic structure optionallyincludes a photonic crystal, a photonic superlattice, a microcavityarray, or an array of plasmonic nanoantennas.

Additionally, or alternatively, the array of features optionallyincludes a plurality of wells. The first feature can include a firstwell within which the first luminophore is disposed, and the secondfeature can include a second well within which the second luminophore isdisposed. Alternatively, the array of features optionally includes aplurality of posts. The first feature can include a first post uponwhich the first luminophore is disposed, and the second feature caninclude a second post upon which the second luminophore is disposed.

Additionally, or alternatively, the first and second characteristicsoptionally are selected independently from the group consisting ofwavelength, polarization, and angle. For example, the firstcharacteristic optionally includes a first linear polarization, and thesecond characteristic optionally includes a second linear polarizationthat is different than the first linear polarization. Optionally, thefirst linear polarization is substantially orthogonal to the secondlinear polarization, or optionally the first linear polarization isrotated relative to the second linear polarization by between about 15degrees and about 75 degrees. Additionally, or alternatively, the firstcharacteristic optionally can include a first wavelength, and the secondcharacteristic optionally can include a second wavelength that isdifferent than the first wavelength.

Additionally, or alternatively, the radiation source optionally includesan optical component. Optionally, the device further includes acontroller coupled to the optical component and configured to controlthe optical component so as to impose the first characteristic on thefirst photons and configured to impose the second characteristic on thesecond photons. Optionally, the optical component includes abirefringent material configured to rotate the first photons to a firstlinear polarization responsive to a first control signal by thecontroller, and configured to rotate the second photons to a secondlinear polarization responsive to a second control signal by thecontroller.

Additionally, or alternatively, the first and second photons optionallyeach irradiate the photonic structure at substantially the same angle asone another. Additionally, or alternatively, the first and secondphotons optionally each irradiate the photonic structure at an angleapproximately normal to a major surface of the photonic structure.Additionally, or alternatively, the first and second photons optionallyeach irradiate the photonic structure at an angle approximately parallelto a major surface of the photonic structure.

Additionally, or alternatively, the second feature optionally islaterally displaced from the first feature.

Additionally, or alternatively, a third feature of the array of featuresoptionally is disposed over the first pixel and spatially displaced fromeach of the first and second features. The device further optionally caninclude a third luminophore disposed within or over the third feature.The radiation source optionally can be configured to generate thirdphotons having a third characteristic at a third time. Optionally, thethird characteristic can be different than the first and secondcharacteristics, and the third time can be different than the first andsecond times. Optionally, the first pixel selectively receivesluminescence emitted by the third luminophore responsive to the thirdphotons at the third time. Additionally, or alternatively, a fourthfeature of the array of features optionally is disposed over the firstpixel and spatially displaced from each of the first, second, and thirdfeatures. The device optionally further includes a fourth luminophoredisposed within or over the fourth feature. The radiation sourceoptionally is configured to generate fourth photons having a fourthcharacteristic at a fourth time. Optionally, the fourth characteristiccan be different than the first, second, and third characteristics, andthe fourth time can be different than the first, second, and thirdtimes. The first pixel optionally selectively receives luminescenceemitted by the fourth luminophore responsive to the fourth photons atthe fourth time. Optionally, the first luminophore is coupled to a firstnucleic acid, the second luminophore is coupled to a second nucleicacid, the third luminophore is coupled to a third nucleic acid, and thefourth luminophore is coupled to a fourth nucleic acid.

Additionally, or alternatively, a third feature of the array of featuresoptionally is disposed over a second pixel of the array of imagingpixels, and a fourth feature of the array of features optionally isdisposed over the second pixel and spatially displaced from the thirdfeature. The device optionally further includes a third luminophoredisposed within or over the third feature, and a fourth luminophoredisposed within or over the fourth feature. Optionally, the second pixelselectively receives luminescence emitted by the third luminophoreresponsive to the first photons at the first time or responsive to thesecond photons at the second time. Optionally, the second pixelselectively receives luminescence emitted by the fourth luminophoreresponsive to the first photons at the first time or responsive to thesecond photons at the second time. Optionally, the first luminophore iscoupled to a first nucleic acid, the second luminophore is coupled to asecond nucleic acid, the third luminophore is coupled to a third nucleicacid, and the fourth luminophore is coupled to a fourth nucleic acid.

Additionally, or alternatively, the first and second features optionallyeach have a substantially circular cross-section. Additionally, oralternatively, the photonic structure optionally includes a hexagonallattice, and optionally the imaging pixels are rectangular.

Additionally, or alternatively, the radiation source optionally isconfigured to flood illuminate the photonic structure with the first andsecond photons. Additionally, or alternatively, the radiation sourceoptionally includes a laser. Additionally, or alternatively, optionallythe first and second photons independently have wavelengths betweenabout 300 nm and about 800 nm.

Additionally, or alternatively, the first luminophore optionally iscoupled to a first nucleic acid, and the second luminophore optionallyis coupled to a second nucleic acid. Additionally, or alternatively, thedevice optionally includes at least one microfluidic feature in contactwith the array of features and configured to provide a flow of one ormore analytes to the first and second features.

Additionally, or alternatively, the first luminophore optionally iscoupled to a first polynucleotide to be sequenced, and the secondluminophore optionally is coupled to a second polynucleotide to besequenced. Optionally, the first polynucleotide is coupled to the firstfeature, and optionally the second polynucleotide is coupled to thesecond feature. Additionally, or alternatively, the device optionallyfurther includes a first polymerase adding a first nucleic acid to athird polynucleotide that is complementary to and coupled to the firstpolynucleotide. The first nucleic acid optionally can be coupled to thefirst luminophore. The device optionally further includes a secondpolymerase adding a second nucleic acid to a fourth polynucleotide thatis complementary to and coupled to the second polynucleotide. The secondnucleic acid optionally can be coupled to the second luminophore.Optionally, the device further can include a channel flowing a firstliquid including the first and second nucleic acids and the first andsecond polymerases into or over the first and second features.

Under another aspect, a method is provided for use in luminescentimaging. The method can include providing an array of imaging pixels,and providing a photonic structure disposed over the array of imagingpixels. The method further can include providing an array of featuresdisposed over the photonic structure. A first feature of the array offeatures can be disposed over a first pixel of the array of imagingpixels, and a second feature of the array of features can be disposedover the first pixel and spatially displaced from the first feature. Themethod further can include providing a first luminophore disposed withinor over the first feature, and providing a second luminophore disposedwithin or over the second feature. The method further can includegenerating by a radiation source first photons having a firstcharacteristic at a first time, and generating by the radiation sourcesecond photons having a second characteristic at a second time. Thesecond characteristic can be different than the first characteristic,and the second time can be different than the first time. The methodfurther can include selectively receiving by the first pixelluminescence emitted by the first luminophore responsive to the firstphotons at the first time; and selectively receiving by the first pixelluminescence emitted by the second luminophore responsive to the secondphotons at the second time.

Optionally, the first photons having the first characteristic generate afirst resonant pattern within the photonic structure at the first time,the first resonant pattern selectively exciting the first luminophorerelative to the second luminophore. Optionally, the second photonshaving the second characteristic generate a second resonant patternwithin the photonic structure at the second time, the second resonantpattern selectively exciting the second luminophore relative to thefirst luminophore.

Additionally, or alternatively, the array of imaging pixels, thephotonic structure, and the array of features optionally aremonolithically integrated with one another.

Additionally, or alternatively, the photonic structure optionallyincludes a photonic crystal, a photonic superlattice, a microcavityarray, or an array of plasmonic nanoantennas.

Additionally, or alternatively, the array of features optionallyincludes a plurality of wells. The first feature optionally can includea first well within which the first luminophore is disposed, and thesecond feature optionally can include a second well within which thesecond luminophore is disposed. Alternatively, the array of features caninclude a plurality of posts. The first feature optionally can include afirst post upon which the first luminophore is disposed, and the secondfeature optionally can include a second post upon which the secondluminophore is disposed.

Additionally, or alternatively, the first and second characteristicsoptionally can be selected independently from the group consisting ofwavelength, polarization, and angle. For example, the firstcharacteristic optionally can include a first linear polarization, andthe second characteristic optionally can include a second linearpolarization that is different than the first linear polarization.Optionally, the first linear polarization can be substantiallyorthogonal to the second linear polarization, or can be rotated relativeto the second linear polarization by between about 15 degrees and about75 degrees. Additionally, or alternatively, the first characteristicoptionally includes a first wavelength, and the second characteristicoptionally includes a second wavelength that is different than the firstwavelength.

Additionally, or alternatively, the radiation source optionally includesan optical component. The method optionally further includes controllingthe optical component so as to impose the first characteristic on thefirst photons and so as to impose the second characteristic on thesecond photons. Optionally, the optical component includes abirefringent material rotating the first photons to a first linearpolarization responsive to a first control signal by a controller, androtating the second photons to a second linear polarization responsiveto a second control signal by the controller.

Additionally, or alternatively, the first and second photons optionallyeach irradiate the photonic structure at substantially the same angle asone another. Additionally, or alternatively, the first and secondphotons optionally each irradiate the photonic structure at an angleapproximately normal to a major surface of the photonic structure, orthe first and second photons optionally each irradiate the photonicstructure at an angle approximately parallel to a major surface of thephotonic structure.

Additionally, or alternatively, the second feature optionally islaterally displaced from the first feature.

Additionally, or alternatively, a third feature of the array of featuresoptionally is disposed over the first pixel and spatially displaced fromeach of the first and second features. Optionally, the method furtherincludes providing a third luminophore disposed within or over the thirdfeature, and generating third photons having a third characteristic at athird time. The third characteristic optionally can be different thanthe first and second characteristics, and the third time optionally canbe different than the first and second times. The method optionallyfurther can include selectively receiving by the first pixelluminescence emitted by the third luminophore responsive to the thirdphotons at the third time. Optionally, a fourth feature of the array offeatures is disposed over the first pixel and spatially displaced fromeach of the first, second, and third features. The method optionallyfurther includes providing a fourth luminophore disposed within or overthe fourth feature, and generating fourth photons having a fourthcharacteristic at a fourth time. The fourth characteristic optionallycan be different than the first, second, and third characteristics, thefourth time optionally can be different than the first, second, andthird times. The method optionally further can include selectivelyreceiving by the first pixel luminescence emitted by the fourthluminophore responsive to the fourth photons at the fourth time.Optionally, the first luminophore is coupled to a first nucleic acid,the second luminophore is coupled to a second nucleic acid, the thirdluminophore is coupled to a third nucleic acid, and the fourthluminophore is coupled to a fourth nucleic acid.

Additionally, or alternatively, a third feature of the array of featuresoptionally can be disposed over a second pixel of the array of imagingpixels, and a fourth feature of the array of features is disposed overthe second pixel and spatially displaced from the third feature. Themethod optionally further includes providing a third luminophoredisposed within or over the third feature, and providing a fourthluminophore disposed within or over the fourth feature. The methodoptionally further includes selectively receiving by the second pixelluminescence emitted by the third luminophore responsive to the firstphotons at the first time or responsive to the second photons at thesecond time; and selectively receiving by the second pixel luminescenceemitted by the fourth luminophore responsive to the first photons at thefirst time or responsive to the second photons at the second time.Optionally, the first luminophore is coupled to a first nucleic acid,the second luminophore is coupled to a second nucleic acid, the thirdluminophore is coupled to a third nucleic acid, and the fourthluminophore is coupled to a fourth nucleic acid.

Additionally, or alternatively, the first and second features optionallyeach have a substantially circular cross-section. Additionally, oralternatively, the photonic structure optionally includes a hexagonallattice, and the imaging pixels optionally are rectangular.

Additionally, or alternatively, the method optionally includes floodilluminating the photonic structure with the first and second photons.Additionally, or alternatively, the method optionally includesgenerating the first and second photons with a laser. Additionally, oralternatively, optionally the first and second photons independentlyhave wavelengths between about 300 nm and about 800 nm.

Additionally, or alternatively, the first luminophore optionally iscoupled to a first nucleic acid, and the second luminophore optionallyis coupled to a second nucleic acid. Additionally, or alternatively, themethod optionally further includes providing at least one microfluidicfeature in contact with the array of features and flowing, by the atleast one microfluidic feature, one or more analytes to the first andsecond features.

Additionally, or alternatively, the first luminophore optionally iscoupled to a first polynucleotide to be sequenced, and the secondluminophore optionally is coupled to a second polynucleotide to besequenced. Optionally, the first polynucleotide is coupled to the firstfeature, and the second polynucleotide optionally is coupled to thesecond feature. Additionally, or alternatively, the method optionallyincludes adding, by a first polymerase, a first nucleic acid to a thirdpolynucleotide that is complementary to and coupled to the firstpolynucleotide. The first nucleic acid optionally can be coupled to thefirst luminophore. The method optionally further includes adding, by asecond polymerase, a second nucleic acid to a fourth polynucleotide thatis complementary to and coupled to the second polynucleotide. The secondnucleic acid optionally can be coupled to the second luminophore.Optionally, the method further includes flowing, by a channel, a firstliquid including the first and second nucleic acids and the first andsecond polymerases into or over the first and second features.

Under another aspect, a device is provided for use in luminescentimaging. The device can include an array of imaging pixels, and aphotonic structure disposed over the array of imaging pixels. The devicefurther can include an array of features disposed over the photonicstructure. A first feature of the array of features can be disposed overa first pixel of the array of imaging pixels, and a second feature ofthe array of features can be disposed over the first pixel and spatiallydisplaced from the first feature. The photonic structure can be tuned toselectively irradiate the first feature with light of a firstpolarization compared to light of a second polarization. The photonicstructure can be tuned to selectively irradiate the second feature withlight of a second polarization compared to light of the firstpolarization.

Optionally, the device further includes a radiation source configured togenerate first photons having the first polarization at a first time,and configured to generate second photons having the second polarizationat a second time.

Additionally, or alternatively, the device optionally further includes afirst luminophore disposed within or over the first feature and a secondluminophore disposed within or over the second feature.

Additionally, or alternatively, the device optionally further includes afirst target analyte disposed within or over the first feature and asecond target analyte disposed within or over the second feature. Thefirst target analyte optionally can be different from the second targetanalyte. the first and second target analytes optionally include nucleicacids having different sequences.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A schematically illustrates a perspective view of an exemplaryprior art photonic structure-based device for use in luminescent imagingof a site within a pixel.

FIG. 1B schematically illustrates a perspective view of an exemplaryprior art array of sites within an array of devices such as illustratedin FIG. 1A, wherein each site corresponds to a pixel.

FIG. 1C schematically illustrates a cross-sectional view of an exemplaryprior art device such as illustrated in FIG. 1A.

FIG. 2A schematically illustrates a perspective view of exemplaryexcitation of the prior art array of sites illustrated in FIG. 1B.

FIG. 2B schematically illustrates simulated exemplary prior art fieldstrengths within an array of devices such as illustrated in FIGS. 1A and1C responsive to excitation such as illustrated in FIG. 2A.

FIG. 3A schematically illustrates a perspective view of an exemplaryarray of sites such as provided herein, wherein multiple sitescorrespond to a pixel.

FIG. 3B schematically illustrates a cross-sectional view of a devicesuch as provided herein, wherein multiple sites correspond to a pixelsuch as illustrated in FIG. 3A.

FIG. 4A schematically illustrates a perspective view of exemplaryexcitation of selected sites of the array of sites illustrated in FIG.3A using scanning focused beam illumination such as provided herein.

FIG. 4B schematically illustrates a perspective view of exemplaryexcitation of selected sites of the array of sites illustrated in FIG.3A using multi-laser interference illumination such as provided herein.

FIG. 5 schematically illustrates an exemplary photonic structure such ascan be included in a device such as provided herein and illustrated inFIGS. 3A-3B.

FIGS. 6A-6D schematically illustrate exemplary simulated field strengthswithin a photonic structure such as illustrated in FIG. 5 , for aradiation source that respectively generates photons having differentcharacteristics than one another at different times.

FIG. 7A schematically illustrates a plan view of an exemplary photonicstructurebased device such as provided herein and illustrated in FIGS.3A-3B that includes first and second sites (e.g., clusters) per pixel.

FIG. 7B schematically illustrates exemplary simulated field strengthswithin an array of devices such as provided herein and illustrated inFIGS. 7A and 3A-3B for a radiation source generating photons having afirst characteristic selectively exciting the first site at a firsttime.

FIG. 7C schematically illustrates exemplary simulated field strengthswithin an array of devices such as provided herein and illustrated inFIGS. 7A and 3A-3B for a radiation source generating photons having asecond characteristic selectively exciting the second site at a secondtime.

FIG. 7D schematically illustrates exemplary cross-talk terms resultingfrom selective excitation of first and second sites such as providedherein and respectively illustrated in FIGS. 7B and 7C.

FIG. 8A schematically illustrates a plan view of an exemplary photonicstructurebased device such as provided herein and illustrated in FIGS.3A-3B that includes first, second, and third sites (e.g., clusters) perpixel.

FIG. 8B schematically illustrates exemplary simulated field strengthswithin an array of devices such as provided herein and illustrated inFIGS. 8A and 3A-3B for a radiation source generating photons having afirst characteristic selectively exciting the first site at a firsttime.

FIG. 8C schematically illustrates exemplary simulated field strengthwithin an array of devices such as provided herein and illustrated inFIGS. 8A and 3A-3B for a radiation source generating photons having asecond characteristic selectively exciting the second site at a secondtime.

FIG. 8D schematically illustrates exemplary simulated field strengthwithin an array of devices such as provided herein and illustrated inFIGS. 8A and 3A-3B for a radiation source generating photons having athird characteristic selectively exciting the third site at a thirdtime.

FIG. 8E schematically illustrates exemplary cross-talk terms resultingfrom selective excitation of first, second, and third sites such asprovided herein and respectively illustrated in FIGS. 8B-8D, accordingto some embodiments.

FIGS. 9A-9D respectively schematically illustrate perspective views ofexemplary selective excitation of first, second, third, and fourth siteswithin an array of devices such as provided herein and illustrated inFIGS. 3A-3B using a radiation source generating photons having differentcharacteristics at different times.

FIG. 10 illustrates an exemplary flow of steps in a method providedherein for use in luminescent imaging.

FIG. 11 illustrates an exemplary sequence of steps that can be used toprepare a device or composition such as provided herein.

FIG. 12 illustrates an exemplary sequence of steps that can be used toprepare a device or composition such as provided herein.

FIG. 13 illustrates an exemplary device for use in luminescent imagingsuch as provided herein.

DETAILED DESCRIPTION

Embodiments of the present invention provide photonic structure-baseddevices and compositions for use in luminescent imaging of multiplesites within a pixel, and methods of using the same.

First, some exemplary terms will be defined, followed by furtherdescription of exemplary embodiments of the present photonicstructure-based devices and compositions for use in luminescent imaging,and methods of using the same.

As used herein, the term “photonic structure” means a periodicstructure, including one or more optically transparent materials, thatselectively affects the propagation of radiation having a particularcharacteristic, e.g., at a wavelength, an angle, and at a polarization.For example, the photonic structure can selectively propagate radiationhaving such characteristic, e.g., at the wavelength, the angle, and thepolarization, through the structure or at the same angle or a differentangle out of the structure, and the field strength of such radiation canhave a selected pattern within the photonic structure. Additionally, thestructure can selectively inhibit propagation of radiation having adifferent characteristic, e.g., at a different wavelength, angle, and/orpolarization, through the structure or at a different angle out of thestructure, and/or the field strength of such radiation can have adifferent selected pattern within the photonic structure. Thematerial(s) of the photonic structure can include features that aredistributed in one or more dimensions, e.g., in one dimension, in twodimensions, or in three dimensions. The shape, size, and distribution ofthe features of the photonic structure, as well as the refractive indexof the material(s), can be tuned so as select the particular radiationcharacteristic(s), e.g., wavelength(s), angle(s), or polarization(s),that can propagate through or at an angle out of the photonic structure,and/or so as to select the pattern of the field strength of suchradiation within the photonic structure. Exemplary photonic structuresinclude, but are not limited to, photonic crystals, photonicsuperlattices, microcavity arrays, and arrays of plasmonic nanoantennas.

As used herein, the terms “photonic crystal,” “PhC,” “photonic lattice,”“photonic crystal lattice,” and “PhC lattice” mean a photonic structureincluding one or more materials that include a periodic variation ofrefractive index on the order of the wavelength of light. For example, aphotonic crystal can include a material that extends in threedimensions, e.g., has a length, a width, and a thickness. The materialcan have two major surfaces that each lie within a plane defined by thelength and the width, and separated from one another by the thickness.The material can be patterned in two or more dimensions so as to definea photonic band structure within which radiation having particularcharacteristic(s), e.g., wavelength(s), angle(s), or polarization(s),can propagate through or at an angle out of the photonic crystal, and/orso as to select the pattern of the field strength of such radiationwithin the photonic crystal. The pattern can include, for example, aplurality of features such as wells or posts that are defined within thematerial, e.g., through one or both of the major surfaces of thematerial, the material being absent within or between the features, suchas within the wells or between the posts. A space within or between thefeatures can be filled with one or more additional materials thatrespectively can have different refractive indices than that of thematerial and than that of one another. The particular characteristic(s)of radiation, e.g., wavelength(s), angle(s), or polarization(s), thatpropagate or do not propagate through, or at an angle out of, thephotonic crystal can be based on the refractive indices of the materialand of any additional materials disposed within the features or betweenthe features, as well as based on the characteristics of the features,such as the shape, size, and distribution of the features. The featurescan be all the same shape, size, and/or distribution as one another.

As used herein, the terms “photonic superlattice” and “PhC superlattice”mean a photonic structure that selectively affects the propagation ofradiation having first and second characteristics, e.g., at first andsecond wavelength(s), angle(s), or polarization(s), compared toradiation having third characteristics, e.g., at a third wavelength,angle, or polarization. For example, the field strength of the radiationhaving the first characteristics can have a first pattern, and the fieldstrength of the radiation having the second characteristics can have asecond pattern that is different from the first pattern. The thirdwavelength can occur between the first and second wavelengths in theelectromagnetic spectrum. For example, the photonic superlattice canselectively propagate radiation having the first and secondcharacteristics, e.g., at the first and second wavelength(s), angle(s),or polarization(s), through the photonic superlattice or at an angle outof the photonic superlattice, and the patterns of the field strengthsfor the radiation having the first and second characteristics optionallycan be different than one another. For example, the photonicsuperlattice can selectively inhibit propagation of radiation having thefirst and second characteristics, e.g., at the first and secondwavelength(s), angle(s), or polarization(s), through the photonicsuperlattice or at an angle out of the photonic superlattice. Forexample, the photonic superlattice can selectively propagate radiationhaving third characteristics, e.g., at the third wavelength, angle, orpolarization, through the photonic superlattice or at an angle out ofthe photonic superlattice. For example, the photonic superlattice canselectively inhibit propagation of radiation having thirdcharacteristics, e.g., at the third wavelength, angle, or polarization,through the structure or at an angle out of the structure. Thematerial(s) can include features that are distributed in one or moredimensions, e.g., in one dimension, in two dimensions, or in threedimensions. The shape, size, and distribution of the features, as wellas the refractive index of the material(s), can be tuned so as selectthe particular characteristics of radiation, e.g., wavelength(s),angle(s), or polarization(s), that can propagate through or at an angleout of the photonic superlattice, as well as the patterns of fieldstrength of such characteristics, and so as to select the particularcharacteristics of radiation that do not propagate substantially throughor at an angle out of the photonic superlattice.

Illustratively, a photonic superlattice can include a material thatextends in three dimensions, e.g., has a length, a width, and athickness. The material can have two major surfaces that each lie withina plane defined by the length and the width, and separated from oneanother by the thickness. The material can be patterned in two or moredimensions so as to define a photonic band structure that permitspropagation of radiation having at least first and secondcharacteristics, e.g., wavelength(s), angle(s), or polarization(s),within, or at an angle out of, the plane defined by the length and thewidth, and that inhibits propagation of at least radiation having thirdcharacteristics, e.g., a third wavelength, angle, or polarization,within, or at an angle out of, the material. The pattern can include,for example, a plurality of features such as wells or posts that aredefined within the material, e.g., through one or both of the majorsurfaces of the material, the material being absent within or betweenthe features, such as within the wells or between the posts. A spacewithin or between the features can be filled with one or more additionalmaterials that respectively can have different refractive indices thanthat of the material and than that of one another. The particularcharacteristics of radiation that propagate or do not propagate through,or at an angle out of, the photonic superlattice can be based on therefractive indices of the material and of any additional materialsdisposed within the features or between the features, as well as basedon the characteristics of the features, such as the shape, size, anddistribution of the features. Some of the features optionally can differin at least one characteristic, e.g., shape, size, or distribution, fromothers of the features. For further details regarding exemplary photonicsuperlattices that can be used in the present devices, compositions, andmethods, see U.S. Provisional Patent Application No. 62/312,704, filedMar. 24, 2016 and entitled “Photonic Superlattice-Based Devices andCompositions for Use in Luminescent Imaging, and Methods of Using theSame,” the entire contents of which are incorporated by referenceherein.

As used herein, “microcavity array” means a periodic two-dimensionalarrangement of photonic microresonators that support multiple (e.g., atleast two, at least three, or at least four) resonances that can beexcited independently of one another by changing a characteristic of anexcitation source, such as the wavelength, polarization, or angle of theexcitation source. For further details regarding exemplary microcavityarrays that can be used in the present devices, compositions, andmethods, see Altug et al., “Polarization control and sensing withtwodimensional coupled photonic crystal microcavity arrays,” Opt. Lett.30: 1422-1428 (2011), the entire contents of which are incorporated byreference herein.

As used herein, “array of plasmonic nanoantennas” means a periodictwodimensional arrangement of plasmonic nanostructures that supportmultiple (e.g., at least two, at least three, or at least four)resonances that can be excited independently of one another by changinga characteristic of an excitation source, such as the wavelength,polarization, or angle of the polarization source. For further detailsregarding exemplary plasmonic nanoantennas that can be used in thepresent devices, compositions, and methods, see Regmi et al., “Nanoscalevolume confinement and fluorescence enhancement with double nanoholeaperture,” Scientific Reports 5: 15852-1-5 (2015), the entire contentsof which are incorporated by reference herein.

One or more of the materials of the photonic structure can be or includea “dielectric material,” meaning a fluidic, solid, or semi-solidmaterial that is optically transparent and is an electrical insulator.Examples of fluidic dielectric materials include gases such as air,nitrogen, and argon, as well as liquids such as such as water, aqueoussolvents, and organic solvents. Examples of solid dielectric materialsinclude glasses (e.g., inorganic glasses such as silica, or modified orfunctionalized glasses) and polymers (such as acrylics, polystyrene,copolymers of styrene and other materials, polypropylene, polyethylene,polybutene, polyurethanes, TEFLON™, cyclic olefins, polyimides, ornylon). Examples of semi-solid dielectric materials include gels, suchas hydrogels. Additionally, or alternatively, one or more materials ofthe photonic structure can be or include a solid semiconductor materialthat is optically transparent.

As used herein, the term “gel” is intended to mean a semi-solid orsemi-rigid material that is permeable to liquids and gases. Typically,gel material can swell when liquid is taken up and can contract whenliquid is removed by drying. Exemplary gels can include, but are notlimited to, those having a colloidal structure, such as agarose or ahydrogel; polymer mesh structure, such as gelatin; or cross-linkedpolymer structure, such as polyacrylamide, SFA (see, for example, US2011/0059865, the entire contents of which are incorporated by referenceherein) or PAZAM (see, for example, US 2014/0079923, the entire contentsof which are incorporated by reference herein). Particularly useful gelmaterial will conform to the shape of a well or other concave featurewhere it resides.

As used herein, the term “well” means a discrete concave feature in amaterial having a surface opening (aperture) that is completelysurrounded by interstitial region(s) of the surface. A well can havecharacteristics such as size (e.g., volume, diameter, and depth),cross-sectional shape (e.g., round, elliptical, triangular, square,polygonal, star shaped (having any suitable number of vertices),irregular, or having concentric wells separated by a dielectricmaterial), and distribution (e.g., spatial locations of the wells withinthe dielectric material, e.g., regularly spaced or periodic locations,or irregularly spaced or aperiodic locations). The cross section of awell can be, but need not necessarily be, uniform along the length ofthe well.

As used herein, the term “post” means a discrete convex featureprotruding from the surface of a material and that is completelysurrounded by interstitial region(s) of the surface. A post can havecharacteristics such as size (e.g., volume, diameter, and depth), shape(e.g., round, elliptical, triangular, square, polygonal, star shaped(having any suitable number of vertices), irregular, or havingconcentric posts separated by a dielectric material), and distribution(e.g., spatial locations of the posts protruding from the surface of thedielectric material, e.g., regularly spaced or periodic locations, orirregularly spaced or aperiodic locations). The cross section of a postcan be, but need not necessarily be, uniform along the length of thepost.

As used herein, the term “surface” means a part or layer of a materialthat is in contact with another material.

As used herein, the term “interstitial region” is intended to mean anarea in a material or on a surface that separates areas of the materialor surface. For example, an interstitial region can separate one featureof a photonic structure from another feature of a photonic structure, oran interstitial region can separate one site of an array from anothersite of the array.

As used herein, the term “luminescent” means emitting cold bodyradiation, and the term “luminophore” means an item that is luminescent.The term “luminescent” is intended to be distinct from incandescencewhich is radiation emitted from a material as a result of heat.Generally luminescence results when an energy source displaces anelectron of an atom out of its lowest energy ground state into a higherenergy excited state; then the electron returns the energy in the formof radiation so it can fall back to its ground state. A particularlyuseful type of luminescent item is one that emits cold body radiationwhen energy is provided by excitation radiation. Such items can bereferred to as “photoluminescent.” Examples of photoluminescent itemsinclude “fluorescent” items that emit cold body radiation relativelyquickly (e.g., less than a millisecond) after excitation radiation, and“phosphorescent” items that emit cold body radiation relatively slowly(e.g., greater than or equal to a millisecond) after excitationradiation. Photoluminescence can be perceived as emission of radiationby an item at a wavelength that is a result of irradiating the item atanother wavelength. Another useful type of luminescent item is one thatemits cold body radiation when energy is provided by a chemical orbiological reaction. Such items can be referred to as“chemiluminescent.”

Any of a variety of signals can be detected in a method set forth hereinincluding, for example, an optical signal such as absorbance ofradiation, luminescence emission, luminescence lifetime, luminescencepolarization, or the like; Rayleigh and/or Mie scattering; or the like.Exemplary labels that can be detected in a method set forth hereininclude, without limitation, a fluorophore, luminophore, chromophore,nanoparticle (e.g., gold, silver, carbon nanotubes), or the like.

As used herein the term “feature” means a distinctive variation in thestructure or composition of a material such as a solid support.Optionally, the variation is also repeated in the structure orcomposition of the material. A collection of the features can form anarray or lattice in or on the material. Exemplary features include, butare not limited to wells, posts, ridges, channels, sites bearinganalytes, layers of a multilayer material, areas in or on a materialhaving a chemical composition that differ from the chemical compositionof other areas in or on the material and the like. A feature can havecharacteristics such as size (e.g., volume, diameter, and depth), shape(e.g., round, elliptical, triangular, square, polygonal, star shaped(having any suitable number of vertices), irregular, or havingconcentric features separated by a dielectric material), anddistribution (e.g., spatial locations of the features within thedielectric material, e.g., regularly spaced or periodic locations, orirregularly spaced or aperiodic locations). The cross section of afeature can be, but need not necessarily be, uniform along the length ofthe feature.

As used herein, the term “site” means a location in an array for aparticular species of molecule or cell (or other analyte). A site cancontain only a single molecule (or cell or other analyte) or it cancontain a population of several molecules (or cells or analytes) of thesame species. In some embodiments, sites are present on a material priorto attaching a particular analyte. In other embodiments the site iscreated by attachment of a molecule or cell (or other analyte) to thematerial. Sites of an array are typically discrete. The discrete sitescan be contiguous or they can have spaces between each other. It will beunderstood that a site is a type of feature. A feature can function as acomponent of a lattice, array or both.

As used herein, the term “array” means a population of sites that can bedifferentiated from each other according to relative location.

As used herein, the term “pitch,” when used in reference to features ofa lattice (e.g. photonic structure) or array, is intended to refer tothe center-to-center spacing for adjacent features of the lattice orarray. A pattern of features can be characterized in terms of averagepitch. The pattern can be ordered such that the coefficient of variationaround the average pitch is small, or the pattern can be random in whichcase the coefficient of variation can be relatively large. In eithercase, the average pitch can be, for example, at least about on the orderof a wavelength of light in one or more regions of the spectrum. Forexample, the pitch can correspond to wavelengths in one or more of thevisible spectrum (about 380-700 nm), UV spectrum (less than about 380 nmto about 10 nm) and IR spectrum (greater than about 700 nm to about 1mm). In a photonic structure, features can have different pitches thanone another in different directions. For example, in a photonicsuperlattice, different types of features can have different pitches andpatterns than one another. For example, the pitch for the features ofone type (e.g. in a first lattice) can differ from the pitch forfeatures of another type (e.g. in a second lattice).

As used herein, the term “random” can be used to refer to the spatialdistribution, e.g., arrangement, of locations on a surface. For example,one or more features (e.g., wells or posts) of a photonic structure or aphotonic superlattice can be randomly spaced such that nearest neighborfeatures, which can be of the same type or different type than oneanother, have variable spacing between each other. Alternatively, thespacing between features of the same type or a different type than oneanother can be ordered, for example, forming a regular pattern such as arectilinear grid or a hexagonal grid.

As used herein, the term “nucleotide” or “nucleic acid” is intended tomean a molecule that includes a sugar and at least one phosphate group,and optionally also includes a nucleobase. A nucleotide that lacks anucleobase can be referred to as “abasic.” Nucleotides includedeoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides,modified ribonucleotides, peptide nucleotides, modified peptidenucleotides, modified phosphate sugar backbone nucleotides, and mixturesthereof. Examples of nucleotides include adenosine monophosphate (AMP),adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidinemonophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate(TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP),cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosinediphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate(UMP), uridine diphosphate (UDP), uridine triphosphate (UTP),deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP),deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP),deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP),reversibly blocked adenosine triphosphate (rbATP), reversibly blockedthymidine triphosphate (rbTTP), reversibly blocked cytidine triphosphate(rbCTP), and reversibly blocked guanosine triphosphate (rbGTP). Forfurther details on reversibly blocked nucleotide triphosphates (rbNTPs),see U.S. Patent Publication No. 2013/0079232, the entire contents ofwhich are incorporated by reference herein.

The term “nucleotide” or “nucleic acid” also is intended to encompassany nucleotide analogue which is a type of nucleotide that includes amodified nucleobase, sugar and/or phosphate moiety. Exemplary modifiednucleobases that can be included in a polynucleotide, whether having anative backbone or analogue structure, include, inosine, xathanine,hypoxathanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine,5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methylguanine, 2-propyl guanine, 2-propyl adenine, 2-thiouracil,2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine,5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine,6-azo thymine, 5-uracil, 4-thiouracil, 8halo adenine or guanine, 8-aminoadenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine orguanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil orcytosine, 7methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine,7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine or thelike. As is known in the art, certain nucleotide analogues cannot becomeincorporated into a polynucleotide, for example, nucleotide analoguessuch as adenosine 5′-phosphosulfate.

As used herein, the term “polynucleotide” refers to a molecule thatincludes a sequence of nucleotides that are bonded to one another.Examples of polynucleotides include deoxyribonucleic acid (DNA),ribonucleic acid (RNA), and analogues thereof. A polynucleotide can be asingle stranded sequence of nucleotides, such as RNA or single strandedDNA, a double stranded sequence of nucleotides, such as double strandedDNA, or can include a mixture of a single stranded and double strandedsequences of nucleotides. Double stranded DNA (dsDNA) includes genomicDNA, and PCR and amplification products. Single stranded DNA (ssDNA) canbe converted to dsDNA and vice-versa. The precise sequence ofnucleotides in a polynucleotide can be known or unknown. The followingare examples of polynucleotides: a gene or gene fragment (for example, aprobe, primer, expressed sequence tag (EST) or serial analysis of geneexpression (SAGE) tag), genomic DNA, genomic DNA fragment, exon, intron,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA,recombinant polynucleotide, synthetic polynucleotide, branchedpolynucleotide, plasmid, vector, isolated DNA of any sequence, isolatedRNA of any sequence, nucleic acid probe, primer or amplified copy of anyof the foregoing.

As used herein, “chemically coupled” is intended to mean an attachmentbetween a first member and a second member. In some embodiments, such anattachment is normally irreversible under the conditions in which theattached members are used. In other embodiments, such an attachment isreversible but persists for at least the period of time in which it isused for one or more steps of an analytical or preparative technique setforth herein (e.g. an analytical step of detecting a subunit of apolymer). Such attachment can be formed via a chemical bond, e.g., via acovalent bond, hydrogen bond, ionic bond, dipole-dipole bond, Londondispersion forces, or any suitable combination thereof. Covalent bondsare only one example of an attachment that suitably can be used tocouple a first member to a second member. Other examples includeduplexes between oligonucleotides, peptide-peptide interactions, andhapten-antibody interactions such as streptavidin-biotin,streptavidin-desthiobiotin, and digoxigenin-antidigoxigenin. In oneembodiment, an attachment can be formed by hybridizing a firstpolynucleotide to a second polynucleotide that inhibits detachment ofthe first polynucleotide from the second polynucleotide. Alternatively,an attachment can be formed using physical or biological interactions,e.g., an interaction between a first protein and a second protein thatinhibits detachment of the first protein from the second protein. Asused herein, a “polymerase” is intended to mean an enzyme having anactive site that assembles polynucleotides by polymerizing nucleotidesinto polynucleotides. A polymerase can bind a primed single strandedpolynucleotide template, and can sequentially add nucleotides to thegrowing primer to form a polynucleotide having a sequence that iscomplementary to that of the template.

As used herein, the term “approximately” or “about” means within 10% ofthe stated value.

Provided herein are compositions and devices that include photonicstructures, such as for single color or multicolor luminescence signalenhancement from analytes (e.g. DNA clusters) in one or more excitationand/or luminescence emission bands, optionally at normal incidence ofexcitation. For example, monolithic integration of photonic andmicrofluidic chips on top of CMOS imaging arrays can be used to reducethe size of, e.g., miniaturize, DNA sequencers. Throughput of CMOS-basedsequencing devices can be limited by the size of imaging pixels. Forexample, relatively large pixel sizes can be useful for providingsufficient signal collection from individual DNA molecules or clustersof identical molecules. Although pixels can be made smaller so as toincrease throughput, such size reduction can reduce full well capacityand can increase cross-talk between pixels, thereby reducing thesignal-to-noise ratio (SNR) of the imaging, and the sequencing. Such anapproach also can increase the cost of fabricating the imaging array,e.g., by increasing the amount of engineering of the imaging array aswell as the integration of such imaging array with photonic and/ormicrofluidic components.

An alternative way of increasing throughout by providing more testingsites per device can involve introducing multiple luminescence sites(e.g., DNA clusters, microarray reaction chambers, or the like) perpixel. For example, in particular embodiments, the present compositions,devices, and methods can image multiple sites, each of which can includea respective analyte, using an imaging pixel by selectively excitingdifferent sites at different times than one another using an excitationsource, and obtaining a respective image at each such time.Illustratively, an array of imaging pixels can be provided, and multiplesites can be disposed over each such imaging pixel. Relative to aconfiguration in which only one site is disposed over a each givenpixel, the present multi-site per pixel configuration can significantlyincrease the number of sites that can be imaged using a given pixelarray. However, if all of the sites disposed over a given imaging pixelwere to be excited simultaneously with one another, the pixel wouldreceive luminescence from each such site simultaneously with oneanother, thus impeding the ability to distinguish between luminescencefrom one such site and luminescence from another such site based on anelectrical signal that the pixel generates responsive to receiving suchluminescence.

Optical techniques such as provided herein can be used so as selectivelyto excite only a single one of the multiple sites disposed over a givenimaging pixel at a given time, so as to obtain an electrical signal fromthat pixel responsive to luminescence just from that site at that time,and subsequently to excite a second one of the multiple sites over thatimaging pixel at a second time, so as to obtain a second electricalsignal from that pixel responsive to luminescence from that second site.As such, the luminescence from the two sites can be distinguished fromone another based on the electrical signals obtained from the imagingpixel at the two times. As such, the present compositions, devices, andmethods can provide luminescent imaging of a greater number of sitesthan the number of pixels in an imaging array, e.g., an integer multiplen of the number of pixels, where n is greater than or equal to 2, or 3,or 4, or 5, or greater than 5.

As provided herein, the different sites disposed over an imaging pixelcan be selectively excited by selectively directing excitation photonsto respective ones of the sites at different times than one another. Forexample, a focused laser beam can be scanned over the different sites atdifferent times than one another so as to selectively excite ones of thedifferent sites at such times, the pixel generating electrical signalsat such time responsive to the luminescence from the particular sitebeing excited. As another example, the sites can be irradiated at afirst time with any suitable number of laser beams that interfere withone another in such a manner as to generate a first optical intensitypattern that selectively excites one of the sites at the first time, andcan be irradiated at a second time with any suitable number of laserbeams that interfere with one another in such a manner as to generate asecond optical intensity pattern that selectively excites another one ofthe sites at the second time. The pixel can generate respectiveelectrical signals at the first and second times responsive toluminescence from the respective sites. As still another example, thesites can be disposed over or within a photonic structure that isdisposed over the imaging pixel. The photonic structure can beconfigured so as selectively to excite one of the sites over the pixelresponsive to irradiation with photons having a first characteristic ata first time, and selectively to excite another one of the sites overthe pixel responsive to irradiation with photons having a secondcharacteristic at a second time. The pixel can generate respectiveelectrical signals at the first and second times responsive toluminescence from the respective sites.

The present photonic structure-based devices, compositions, and methodsare compatible with previously known epifluorescence microscopy andmicroscope scanning systems (such as those in commercially availablesequencing platforms such as produced by Illumina, Inc. (San Diego,Calif.)) that, in some circumstances, can use multiple fluorescent dyesexcited at normal and imaged at normal incidence in various spectralwindows. Such dyes can be coupled to nucleotides so as to facilitatesequencing polynucleotides such as DNA. However, it should beappreciated that the present photonic structure-based devices,compositions, and methods suitably can be used in any type ofluminescent imaging or any other suitable application, and are notlimited to use in sequencing polynucleotides such as DNA.

Patterning of dielectric substrates previously has been employedsuccessfully to control the size and uniformity of polynucleotideclusters, and to increase the density of such clusters so as to improvethroughput of sequencing. See for example, US Pat. App. Publ. No.2014/0243224 A1, which is incorporated herein by reference. However,reduction in cluster size has resulted in a considerable reduction inthe amount of collected multicolor fluorescence signal. For example,detection of weak multicolor fluorescence signals from large samplingareas can become increasingly difficult as the number of labelednucleotides in DNA clusters is reduced (e.g., down to single-moleculelevels or the resolution limits of the imaging system). Significantfluorescence signal enhancement therefore can be helpful to facilitatenucleotide identification and increase the throughput of next generationSBS systems.

For example, periodic patterning of materials, such as high-indexdielectrics, in the proximity of fluorescently marked biomolecules canenhance fluorescence signal by creating one- or two-dimensionalwaveguides with a periodic variation of the refractive index in on theorder of wavelength of light. Such waveguides, which can be referred toas photonic crystals (PhCs), photonic lattices, photonic crystallattices, or PhC lattices, can support high-Q resonant modes that canboost fluorescent signals by resonantly enhancing fluorophoreexcitation, fluorescence collection, or both. For examples of use ofsingle-color fluorescence signal enhancement using PhC lattices, see thefollowing references, the entire contents of each of which areincorporated by reference herein: U.S. Pat. No. 7,768,640 to Cunninghamet al.; Estrada et al., “Small volume excitation and enhancement of dyefluorescence on a 2D photonic crystal surface,” Opt. Express 18:3693-3699 (2010); Zhen et al., “Enabling enhanced emission andlow-threshold lasing of organic molecules using special Fano resonancesof macroscopic photonic crystals,” PNAS 110: 13711-13716 (2013); Kaji etal., “Fabrication of two-dimensional Ta₂O₅ photonic crystal slabs withultra-low background emission toward highly sensitive fluorescencespectroscopy,” Opt. Express 19: 1422-1428 (2011); and Pokhriyal et al.,“Photonic crystal enhanced fluorescence using a quartz substrate toreduce limits of detection,” Opt. Express 18: 24793-24808 (2010).

PhC lattices also can be used in multicolor fluorescence signalenhancement. For example, dual-excitation fluorescence signal boost hasbeen achieved with PhCs by resonant enhancement of excitation atdifferent wavelengths that requires adjustment of the angle of incidenceof excitation source to match the resonances supported by the PhC. Forfurther details, see U.S. Pat. No. 8,344,333 to Lu et al., the entirecontents of which are incorporated by reference herein. However, becausethe signal enhancement scheme described in Lu et al. operates intrans-fluorescence mode by tuning the illumination angles, such a schemeis not convenient for imaging or sequencing platforms that rely onmulticolor epi-illumination at a fixed angle of incidence for allwavelengths of interest, e.g., a normal, or close to normal, angle ofincidence.

FIG. 1A schematically illustrates a perspective view of an exemplaryphotonic structure-based device for use in luminescent imaging of a sitewithin a pixel. The device illustrated in FIG. 1A includes an imagingpixel, such as a complementary metal oxide semiconductor (CMOS) basedimage sensor; a photonic structure such as a PhC layer disposed over theimaging pixel; and a nanowell defined within a third material disposedover the PhC layer. The PhC layer can include a first material (shown inblack) having a refractive index of n₁, and regular patterns ofuniformly shaped and sized wells that are defined within the firstmaterial and are filled with a second material (shown in white) having arefractive index of n₂, where n₁ and n₂ are different than each other. Asite including one or more luminophores, e.g., one or more analytesrespectively coupled to luminophores, e.g., one or more nucleotidesrespectively coupled to luminophores, can be disposed within thenanowell. The luminophore(s) can be disposed in the near field of thePhC layer and excited evanescently by the excitation wavelengths, e.g.,photons having suitable characteristics (illustrated as largedownward-pointing arrow in FIG. 1A). The imaging pixel can be suitablyelectronically coupled to a detection circuit (not specificallyillustrated), which can be configured so as to receive and analyze anelectrical signal generated by the imaging pixel responsive toluminescence generated by the luminophore(s). Although the imaging pixelis illustrated in FIG. 1A as having a dimension of 1.75 μm on each side,it should be appreciated that imaging pixels of any suitable dimensionscan be used.

Optionally, an array of any suitable number of such devices can beprovided. For example, FIG. 1B schematically illustrates a perspectiveview of an exemplary array of sites within an array of devices such asillustrated in FIG. 1A, wherein each site (represented as a blackcircle) corresponds to a pixel (represented as a rectangle). That is,the exemplary array illustrated in FIG. 1B includes one site for eachpixel. Additionally, each such device can include any suitable numberand type of materials. For example, FIG. 1C schematically illustrates across-sectional view of an exemplary device such as illustrated in FIG.1A. In an exemplary embodiment of the device illustrated in FIGS. 1A-1C,a PhC layer can be disposed over any suitable imaging pixel such asknown in the art. The PhC layer can include a first material, such assilicon nitride (SiN), that is patterned so as to define a photoniccrystal. A second material, such as tantalum oxide (TaO), can bedisposed over the PhC layer. The nanowell can be defined in a thirdmaterial, such as SiN, and a fourth material such as TaO can be disposedover the nanowell. As illustrated in FIGS. 1A-1C, a single nanowell canbe disposed over each imaging pixel. As such, each imaging pixel canreceive luminescence from luminophore(s) disposed within the nanowellover that pixel, and generate a suitable electronic signal responsive toreceipt of such luminescence.

For example, FIG. 2A schematically illustrates a perspective view ofexemplary excitation of the array of sites illustrated in FIG. 1B.Illustratively, the array of sites can be irradiated with uniform(flat-top) illumination from a single optical source, such as a laser.Such irradiation suitably can excite one or more resonant modes withinthe PhC beneath such sites (such as shown in FIGS. 1A and 1C). Forexample, FIG. 2B schematically illustrates simulated exemplary fieldstrengths within an array of devices such as illustrated in FIGS. 1A and1C responsive to excitation such as illustrated in FIG. 2A. The featuresof the PhC can be tuned so as provide a relatively high field strengthin a location disposed immediately beneath nanowell 200, thusselectively exciting luminophore(s) at the site disposed within thatnanowell.

As provided herein, the number of sites can be increased as an integermultiple n>1 of the number of imaging pixels by selectively excitingdifferent ones of such sites at different times than one another. Forexample, FIG. 3A schematically illustrates a perspective view of anexemplary array of sites such as provided herein, wherein multiple sitescorrespond to a pixel. In the non-limiting example illustrated in FIG.3A, four sites (respectively illustrated as circles having differentfills than one another) are provided per pixel (represented asrectangles), although it should be appreciated that any suitable numberof sites can be provided per pixel, e.g., 2 or more sites per pixel, 3or more sites per pixel, 4 or more sites per pixel, or 5 or more sitesper pixel. Such sites can be provided using any suitable features. Forexample, FIG. 3B schematically illustrates a cross-sectional view of adevice such as provided herein, wherein multiple sites correspond to apixel such as illustrated in FIG. 3A. In an exemplary embodiment of thedevice illustrated in FIGS. 3A-3B, an optional photonic structure can bedisposed over any suitable imaging pixel such as known in the art. Theoptional photonic crystal can include a first material, such as siliconnitride (SiN), that is disposed over the imaging pixel; a secondmaterial, such as silicon dioxide (SiO₂) that is disposed over the firstmaterial; and a photonic crystal including a pattern of a thirdmaterial, such as SiN, and a fourth material, such as SiO₂. A fifthmaterial, such as tantalum oxide (TaO), can be disposed over the PhClayer. A plurality of features, such as a plurality of nanowells, can bedefined in a sixth material, such as SiO₂, and a seventh material suchas TaO can be disposed over the plurality of nanowells. As illustratedin FIGS. 3A-3B, multiple features, e.g., multiple nanowells, can bedisposed over each imaging pixel. As such, each imaging pixel canreceive luminescence at different times from luminophore(s) disposedwithin or over each such feature, e.g., within each such nanowell, overthat pixel, and generate a suitable electronic signal responsive toreceipt of such luminescence at such different times. The imaging pixel,the optional photonic structure, and the features optionally can bemonolithically integrated with one another.

It should be appreciated that the optional photonic structureillustrated in FIG. 3B is intended to be exemplary, and not limiting.For example, the photonic structure can include a photonic crystal, or aphotonic superlattice, or a microcavity array, or an array of plasmonicnanoantennas.

Sites such as provided herein, e.g., with reference to FIGS. 3A-3B,selectively can be excited using any suitable technique. For example, aphotonic structure optionally can be omitted, and the sites over a givenpixel can be selectively excited by directing photons to such sites.Illustratively, a focused laser beam can be scanned over the differentsites at different times than one another so as to selectively exciteones of the different sites at such times, the pixel generatingelectrical signals at such time responsive to the luminescence from theparticular site being excited. For example, FIG. 4A schematicallyillustrates a perspective view of exemplary excitation of selected sitesof the array of sites illustrated in FIG. 3A using scanning focused beamillumination such as provided herein. Illustratively, precise control ofexcitation beams can be achieved using high-precision free-space beamsteering, or by sample manipulation, in a manner similar to thatdescribed in Hahn et al., “Laser scanning lithography for surfacemicropatterning on hydrogels,” Adv. Mater. 17: 2939-2942 (2005) or thatdescribed in Brakenhoff et al., “Confocal light scanning microscopy withhigh-aperture immersion lenses,” J. Microsc. 117: 219-232 (1997), theentire contents of both of which are incorporated by reference herein.

As another example, the sites can be irradiated at a first time with anysuitable number of laser beams that interfere with one another in such amanner as to generate a first optical intensity pattern that selectivelyexcites one of the sites at the first time, and can be irradiated at asecond time with any suitable number of laser beams that interfere withone another in such a manner as to generate a second optical intensitypattern that selectively excites another one of the sites at the secondtime. The pixel can generate respective electrical signals at the firstand second times responsive to luminescence from the respective sites.For example, FIG. 4B schematically illustrates a perspective view ofexemplary excitation of selected sites of the array of sites illustratedin FIG. 3A using multi-laser interference illumination such as providedherein. The sites selectively can be irradiated using multi-laserinterference illumination similar to that described in van Wolferen etal., “Laser interference lithography,” in Lithography: Principles,Processes and Materials, pages 133-148, Theodore Hennessy, Ed., NovaScience Publishers, Inc. (2011), or in He et al., “Polarization controlin flexible interference lithography for nano-patterning of differentphotonic structures with optimized contrast,” Optics Express 11518-11525(May 4, 2015), the entire contents of each of which are incorporated byreference herein.

As still another example, the sites can be disposed over or within aphotonic structure that is disposed over the imaging pixel. The photonicstructure can be configured so as selectively to excite one of the sitesover the pixel responsive to irradiation with photons having a firstcharacteristic at a first time, and selectively to excite another one ofthe sites over the pixel responsive to irradiation with photons having asecond characteristic at a second time. The pixel can generaterespective electrical signals at the first and second times responsiveto luminescence from the respective sites. For example, FIG. 5schematically illustrates an exemplary photonic structure such as can beincluded in a device such as provided herein and illustrated in FIGS.3A3B. In the particular embodiment illustrated in FIG. 5 , the photonicstructure can include a photonic crystal (PhC), but it should beappreciated that the photonic structure can include a photonicsuperlattice, or a microcavity array, or an array of plasmonicnanoantennas. The exemplary PhC illustrated in FIG. 5 includes ahexagonal array of features defined within a material, where the spacingΛ_(PhC) between the features is on the order of the wavelength ofexcitation light λ_(excitation).

The photonic structure, e.g., PhC, can be tuned (e.g., the features ofthe PhC can be selected) such that photons having differentcharacteristics than one another can selectively excite differentresonances within the PhC. Photonic structure design parameters can becomputationally adjusted so as to tune resonances to respective desiredlocations and/or excitation or emission peaks of luminophores, forexample using one or more of Finite-Difference Time-Domain (FDTD),Rigorous Coupled-Wave Analysis (RCWA), and Plane-Wave Expansion (PWE).Design optimization can employ multi-parameter sweeps orselfoptimization algorithms to maximize luminescence signal, e.g.,fluorescence signal, in desired physical regions and/or spectralregions. For example, the refractive indices of material(s) that aphotonic structure is to include, the spatial locations at which highfield intensity is desired, and the wavelengths for which it is desiredthat the photonic structure selectively support resonances, can becomputationally defined, and any suitable combination of FDTD, RCWA,PWE, or any other suitable optimization program(s) can be used so as toadjust other parameters of the structure, such as the size, shape, anddistribution of features within the structure, so as to explore thedesign parameter space of the structure and to identify combinations ofparameters that align the spectral and spatial features of the structurewith the desired luminophore location and/or excitation or emissionwavelengths.

For example, FIGS. 6A-6D schematically illustrate exemplary simulatedfield strengths within a photonic crystal such as illustrated in FIG. 5, for a radiation source that respectively generates photons havingdifferent characteristics than one another at different times. Thesimulated photonic crystal included a hexagonal array of air holes in aTa₂O₅ film positioned on top of an SiO₂ substrate. More specifically,FIG. 6A illustrates simulated field strengths within the exemplaryphotonic crystal of FIG. 5 for photons having a first polarization at afirst time, e.g., X-polarization; FIG. 6B illustrates simulated fieldstrengths within that photonic crystal for photons having a secondpolarization at a second time, e.g., Y-polarization; FIG. 6C illustratessimulated field strengths within that photonic crystal for photonshaving a third polarization at a third time, e.g., RX-polarization; andFIG. 6D illustrates simulated field strengths within that photoniccrystal for photons having a fourth polarization at a fourth time, e.g.,RY-polarization. Based on FIGS. 6A-6D, it can be understood that byvarying the polarization of the photons, different patterns of fieldstrengths within the photonic crystal can be excited. It should beappreciated that by different patterns of field strengths varying othercharacteristics of the photons, such as the wavelength or angle of thephotons, different patterns of field strengths within the photoniccrystal similarly can be excited. Similar differences in patterns offield strengths for any suitable types of photonic structures, such asphotonic crystals, photonic superlattices, microcavity arrays, or arraysof plasmonic nanoantennas, similarly can be obtained by suitably tuningthe features of the photonic structures and suitably varying thecharacteristics of photons irradiating that structure at different timesthan one another.

In some embodiments, the present devices, compositions, and methods canprovide multiple luminophore-including sites that respectively spatiallyoverlap with different patterns of field strengths that are excited atdifferent times than one another. For example, FIG. 7A schematicallyillustrates a plan view of an exemplary photonic structure-based devicesuch as provided herein and illustrated in FIGS. 3A-3B that includesfirst and second sites (e.g., clusters) per pixel. The device caninclude an array of imaging pixels, a photonic structure disposed overthe array of imaging pixels; and an array of features disposed over thephotonic structure. The photonic structure can, for example, include aphotonic crystal, a photonic superlattice, a microcavity array, or anarray of plasmonic nanoantennas. The array of imaging pixels, thephotonic structure, and the array of features optionally can bemonolithically integrated with one another. In one nonlimiting example,the photonic structure can include a hexagonal lattice, and the imagingpixels can be rectangular.

A first feature of the array of features can be disposed over a firstpixel of the array of imaging pixels, and a second feature of the arrayof features can be disposed over the first pixel and spatially displacedfrom the first feature. For example, in the non-limiting exampleillustrated in FIG. 7A, a first feature (referred to as “Cluster 1”) anda second feature (referred to as “Cluster 2”) both are disposed over thesame pixel as one another. The second feature can be laterally displacedfrom the first feature in a manner such as illustrated in FIG. 7A. Inone example, the first and second features respectively are positionedat the bottom-right and top-left corners of the metal light-shieldaperture above the pixel, respectively. A first luminophore can bedisposed within or over the first feature, and a second luminophore canbe disposed within or over the second feature. For example, in someembodiments, the array of features can include a plurality of wells; thefirst feature can include a first well within which the firstluminophore is disposed, and the second feature can include a secondwell within which the second luminophore is disposed, e.g., in a mannersimilar to that illustrated in FIG. 3B. In other embodiments, the arrayof features can include a plurality of posts; the first feature caninclude a first post upon which the first luminophore is disposed, andthe second feature can include a second post upon which the secondluminophore is disposed. Illustratively, the first and second features(e.g., wells or posts) each can have a substantially circularcross-section.

The device further can include a radiation source configured to generatefirst photons having a first characteristic at a first time, andconfigured to generate second photons having a second characteristic ata second time, the second characteristic being different than the firstcharacteristic, the second time being different than the first time. Incontrast to embodiments such as described herein with reference to FIGS.4A-4B, the radiation source need not necessarily be configured so as toselectively direct radiation to different sites at different times.Instead, in some embodiments, the radiation source can be configured soas respectively to flood illuminate the photonic structure with thefirst and second photons at the first and second times, and the featuresof the photonic structure selectively can direct radiation to thedifferent sites. Additionally, or alternatively, the radiation sourcecan include a laser. Optionally, the first and second photons emitted bythe radiation source can be in the optical range of the spectrum, e.g.,the first and second photons independently have wavelengths betweenabout 300 nm and about 800 nm.

In some embodiments, the photonic structure can be tuned so as toselectively irradiate the first feature with light of a firstpolarization compared to light of a second polarization, and can betuned to selectively irradiate the second feature with light of a secondpolarization compared to light of the first polarization. For example,the device can include a first luminophore disposed within or over thefirst feature and a second luminophore disposed within or over thesecond feature. Illustratively, the device can include a first targetanalyte disposed within or over the first feature and a second targetanalyte disposed within or over the second feature, wherein the firsttarget analyte is different from the second target analyte. Optionally,the first and second target analytes can include nucleic acids havingdifferent sequences.

In some embodiments, the first pixel can selectively receiveluminescence emitted by the first luminophore responsive to the firstphotons at the first time, and can selectively receive luminescenceemitted by the second luminophore responsive to the second photons atthe second time. For example, the first photons having the firstcharacteristic can generate a first resonant pattern within the photonicstructure at the first time, the first resonant pattern selectivelyexciting the first luminophore relative to the second luminophore.Illustratively, FIG. 7B schematically illustrates exemplary simulatedfield strengths within an array of devices such as provided herein andillustrated in FIGS. 7A and 3A-3B for a radiation source generatingphotons having a first characteristic selectively exciting the firstsite at a first time. It can be seen that the photons having the firstcharacteristic generate a spatial pattern of field strengths that issignificantly more intense at the first feature than at the secondfeature, and thus can selectively excite the first luminophore relativeto the second luminophore at the first time. As such, the imaging pixelcan generate an electrical signal at the first time that substantiallycorresponds to selective excitation of the first luminophore disposedwithin or over the first feature.

Additionally, the second photons having the second characteristic cangenerate a second resonant pattern within the photonic structure at thesecond time, the second resonant pattern selectively exciting the secondluminophore relative to the first luminophore. Illustratively, FIG. 7Cschematically illustrates exemplary simulated field strengths within anarray of devices such as provided herein and illustrated in FIGS. 7A and3A-3B for a radiation source generating photons having a secondcharacteristic selectively exciting the second site at a second time. Itcan be seen that the photons having the second characteristic generate aspatial pattern of field strengths that is significantly more intense atthe second feature than at the first feature, and thus can selectivelyexcite the second luminophore relative to the first luminophore at thesecond time. As such, the imaging pixel can generate an electricalsignal at the second time that substantially corresponds to selectiveexcitation of the second luminophore disposed within or over the secondfeature. Accordingly, two or more luminophores that are within thedetection zone of a particular pixel can be distinguished from eachother using spatial patterns of excitation light applied to theluminophores at different times. This combination of spatial andtemporal separation of excitation events can allow the pixel todistinguish the two or more luminophores within its detection zone.

Note that although the first luminophore can be excited selectivelyrelative to the second luminophore at the first time such as illustratedin FIG. 7B, the second luminophore nonetheless can be excited at thefirst time, to a smaller extent than is the first luminophore.Similarly, although the second luminophore can be excited selectivelyrelative to the first luminophore at the second time such as illustratedin FIG. 7C, the first luminophore nonetheless can be excited at thesecond time, to a smaller extent than is the second luminophore. Suchexcitation of the second luminophore at the first time and of the firstluminophore at the second time can be referred to as “cross-talk.” FIG.7D schematically illustrates exemplary cross-talk terms resulting fromselective excitation of first and second sites such as provided hereinand respectively illustrated in FIGS. 7B and 7C. The photonic structureand/or the respective characteristics of the first and second photonscan be tuned so as to reduce cross-talk to a level at which respectiveluminescence from the first and second luminophores suitably can bedistinguished from one another.

In embodiments such as illustrated in FIGS. 7B and 7C, the first andsecond characteristics of the first and second photons can be selectedindependently from the group consisting of wavelength, polarization, andangle. Illustratively, the first characteristic can include a firstlinear polarization, and the second characteristic can include a secondlinear polarization that is different than the first linearpolarization. As one example, the first linear polarization can besubstantially orthogonal to the second linear polarization.Illustratively, the pattern of field strengths illustrated in FIG. 7B,which selectively excites the first luminophore within or over the firstfeature, is generated using photons having a first linear polarization,such as X-polarization; and the pattern of field strengths illustratedin FIG. 7C, which selectively excites the second luminophore within orover the second feature, is generated using photons having a secondlinear polarization that is substantially orthogonal to the first linearpolarization, such as Y-polarization. However, it should be appreciatedthat the photons at the first and second times can have any suitablepolarizations. For example, the first linear polarization can be rotatedrelative to the second linear polarization by between about 15 degreesand about 75 degrees, e.g., so as to generate other patterns of fieldstrengths such as described herein with reference to FIGS. 6A-6D. Asanother example, the polarization axes can be rotated clockwise by 30degrees and rotated X- and Y-polarized beams (RX- and RY-polarizedbeams) can be used. Additionally, it should be appreciated that thephotons at the first and second times respectively can have any othersuitable characteristics. For example, the first characteristic of thephotons at the first time can include a first wavelength, and the secondcharacteristic of the photons at the second time can include a secondwavelength that is different than the first wavelength.

The characteristics of the first and second photons generated at thefirst and second times can be controlled in any suitable manner. Forexample, in some embodiments, the radiation source of the device caninclude an optical component and a controller coupled to the opticalcomponent. The controller suitably can be configured to control theoptical component so as to impose the first characteristic on the firstphotons and configured to impose the second characteristic on the secondphotons. For example, in embodiments where the respective photoncharacteristics include polarization, the optical component can includea birefringent material configured to rotate the first photons to afirst linear polarization responsive to a first control signal by thecontroller, and configured to rotate the second photons to a secondlinear polarization responsive to a second control signal by thecontroller. In embodiments where the respective photon characteristicsinclude wavelength, the optical component can include an electronicallyadjustable filter disposed in the path of the photons that can beadjusted so as to control the wavelength of photons arriving at thephotonic structure responsive to control signals by the controller, orcan include a portion of the radiation source that can be adjusted so asto control the wavelength of photons being generated at a given time bythe radiation source responsive to control signals by the controller. Inembodiments where the respective photon characteristics include angle,the optical component can include a reflective or transmissive optic,such as a lens and/or mirror, that can be adjusted so as to control theangle of photons arriving at the photonic structure responsive tocontrol signals by the controller. It should be appreciated that morethan one photon characteristic can be varied at a time. For example, anysuitable combination of two or more of the wavelength, angle, andpolarization of the photons can be adjusted so as selectively to excitea given luminophore disposed over a pixel relative to anotherluminophore disposed over that pixel.

In some embodiments, the first and second photons can irradiate thephotonic structure at any suitable angle. For example, the first andsecond photons each can irradiate the photonic structure atsubstantially the same angle as one another, illustratively at an angleapproximately normal to a major surface of the photonic structure, or atan angle approximately parallel to a major surface of the photonicstructure.

In embodiments such as illustrated in FIGS. 7A-7D, it should beappreciated that other features of the array of features can be disposedover other pixels. For example, a third feature of the array of featurescan be disposed over a second pixel of the array of imaging pixels, anda fourth feature of the array of features can be disposed over thesecond pixel and spatially displaced from the third feature. The devicefurther can include a third luminophore disposed within or over thethird feature, and a fourth luminophore disposed within or over thefourth feature. The second pixel can selectively receive luminescenceemitted by the third luminophore responsive to the first photons at thefirst time or responsive to the second photons at the second time, forexample, if the third luminophore can be excited by the first photons orby the second photons. The second pixel selectively can receiveluminescence emitted by the fourth luminophore responsive to the firstphotons at the first time or responsive to the second photons at thesecond time, for example, if the fourth luminophore can be excited bythe first photons or by the second photons.

It also should be appreciated that any suitable number of sites can beprovided per pixel. Illustratively, a device such as illustrated inFIGS. 3A-3B and 7A-7D optionally further can include a third feature ofthe array of features that is disposed over the first pixel andspatially displaced from each of the first and second features. Thedevice can include a third luminophore disposed within or over the thirdfeature. The radiation source can be configured to generate thirdphotons having a third characteristic at a third time, the thirdcharacteristic being different than the first and secondcharacteristics, the third time being different than the first andsecond times. The first pixel can selectively receive luminescenceemitted by the third luminophore responsive to the third photons at thethird time. For example, FIG. 8A schematically illustrates a plan viewof an exemplary photonic structure-based device such as provided hereinand illustrated in FIGS. 3A-3B that includes first, second, and thirdsites (e.g., clusters) per pixel (represented as circles). In a mannersimilar to that described above with reference to FIGS. 6A-6D and 7A-7C,the photonic structure respectively can be irradiated with photonshaving first, second, and third characteristics at first, second, andthird times, so as respectively to excite the first, second, and thirdluminophores at the first, second, and third sites at such times.

For example, FIG. 8B schematically illustrates exemplary simulated fieldstrengths within an array of devices such as provided herein andillustrated in FIGS. 8A and 3A-3B for a radiation source generatingphotons having a first characteristic selectively exciting the firstsite at a first time. FIG. 8C schematically illustrates exemplarysimulated field strength within such an array of devices for a radiationsource generating photons having a second characteristic selectivelyexciting the second site at a second time. FIG. 8D schematicallyillustrates exemplary simulated field strength within such an array ofdevices a radiation source generating photons having a thirdcharacteristic selectively exciting the third site at a third time. Asone example, the first characteristic can include a first linearpolarization, e.g., Y-polarization, the second characteristic caninclude a second linear polarization, e.g., RY-polarization, and thethird characteristic can include a third linear polarization, e.g.,RX-polarization. Note that one or more of such polarizations can be, butneed not necessarily be, orthogonal to one another. For example, the RX-and RY-polarizations are orthogonal to one another, and each are at anangle between about 15 degrees and about 75 degrees to theY-polarization, e.g., 45 degrees. For example, the first linearpolarization can be rotated relative to the second linear polarizationby between about 15 degrees and about 75 degrees, e.g., so as togenerate other patterns of field strengths such as described herein withreference to FIGS. 6A-6D. As another example, the polarization axes canbe rotated clockwise by 30 degrees and rotated X- and Y-polarized beams(RX- and RY-polarized beams) can be used. Additionally, note that in amanner similar to that described above with reference to FIG. 7D,selectively exciting the first site at the first time also may excitethe second and/or third sites to a lesser degree, selectively excitingthe second site at the second time also may excite the first and/orthird sites, and/or selectively exciting the third site at the thirdtime also may excite the first and/or second sites. FIG. 8Eschematically illustrates exemplary cross-talk terms resulting fromselective excitation of first, second, and third sites such as providedherein and respectively illustrated in FIGS. 8B-8D, according to someembodiments. The photonic structure and/or the respectivecharacteristics of the first and second photons can be tuned so as toreduce cross-talk to a level at which respective luminescence from thefirst and second luminophores suitably can be distinguished from oneanother.

The present devices suitably further can include an even greater numberof sites disposed over each pixel. For example, the device such asdescribed above with reference to FIGS. 3A-3B and 8A-8E optionallyfurther can include a fourth feature of the array of features that isdisposed over the first pixel and spatially displaced from each of thefirst, second, and third features. The device further can include afourth luminophore disposed within or over the fourth feature. Theradiation source can be configured to generate fourth photons having afourth characteristic at a fourth time, the fourth characteristic beingdifferent than the first, second, and third characteristics, the fourthtime being different than the first, second, and third times. The firstpixel can selectively receive luminescence emitted by the fourthluminophore responsive to the fourth photons at the fourth time.Illustratively, FIGS. 9A-9D respectively schematically illustrateperspective views of exemplary selective excitation of first, second,third, and fourth sites within an array of devices such as providedherein and illustrated in FIGS. 3A-3B using a radiation sourcegenerating photons having different characteristics at different times.For example, in a manner such as illustrated in FIG. 9A, at a first timethe photonic structure can be irradiated with photons having a firstcharacteristic (e.g., a first polarization, such as Xpolarization) so asselectively to excite a first site disposed over each pixel.Subsequently, in a manner such as illustrated in FIG. 9B, at a secondtime the photonic structure can be irradiated with photons having asecond characteristic (e.g., a second polarization, such asXYpolarization) so as selectively to excite a second site disposed overeach pixel. Subsequently, in a manner such as illustrated in FIG. 9C, ata third time the photonic structure can be irradiated with photonshaving a third characteristic (e.g., a third polarization, such asYX-polarization) so as selectively to excite a third site disposed overeach pixel. Subsequently, in a manner such as illustrated in FIG. 9D, ata fourth time the photonic structure can be irradiated with photonshaving a fourth characteristic (e.g., a fourth polarization, such asY-polarization). The pixels respectively can generate electrical signalsat the first, second, third, and fourth times, based upon which thefirst, second, third, and fourth sites disposed over such pixels can bedistinguished from one another.

The present compositions, devices, and methods suitably can be used soas to generate luminescent images in SBS sequencing fluorescence signalenhancement at normal incidence illumination. For example, the devicefurther can include at least one microfluidic feature in contact withthe array of features and configured to provide a flow of one or moreanalytes to the first and second features. Additionally, oralternatively, the present compositions, devices, and methods canenhance excitation efficiency of any suitable number of luminophoresusing any suitable number of excitation wavelengths, e.g., can enhanceexcitation efficiency of four distinct excitation sources at fourresonant wavelengths (□₁, □₂, □₃, and □₄) in a 4-channel SBS chemistryscheme, or can enhance excitation efficiency at two excitationwavelengths (□₁ and □₂) in a 2-channel SBS chemistry scheme, or canenhance excitation efficiency at one excitation wavelength (□₁) in a1-channel SBS chemistry scheme. Exemplary 4-channel, 3channel, 2-channelor 1-channel SBS schemes are described, for example, in US Pat. App.Pub. No. 2013/0079232 A1 (incorporated herein by reference) and can bemodified for use with the apparatus and methods set forth herein. Forexample, referring again to embodiments such as described with referenceto FIGS. 7A-7D in which first and second luminophores are disposed overa first pixel, the first luminophore can be coupled to a first nucleicacid, and the second luminophore can be coupled to a second nucleicacid. As another example, referring to the optional embodiment describedwith reference to FIGS. 7A-7D in which first and second luminophores aredisposed over a first pixel and third and fourth luminophores aredisposed over a second pixel, the first luminophore can be coupled to afirst nucleic acid, the second luminophore can be coupled to a secondnucleic acid, the third luminophore is coupled to a third nucleic acid,and the fourth luminophore can be coupled to a fourth nucleic acid. Asstill another example, referring again to the illustrative embodimentsdescribed with reference to FIGS. 9A9D, the first luminophore can becoupled to a first nucleic acid, the second luminophore can be coupledto a second nucleic acid, the third luminophore can be coupled to athird nucleic acid, and the fourth luminophore can be coupled to afourth nucleic acid. For example, in compositions for use in sequencingDNA using luminescent imaging, the first luminophore can be coupled toA, the second luminophore can be coupled to G, the third luminophore canbe coupled to C, and the fourth luminophore can be coupled to T. Asanother example, in compositions for use in sequencing RNA usingluminescent imaging, the first luminophore can be coupled to A, thesecond luminophore can be coupled to G, the third luminophore can becoupled to C, and the fourth luminophore can be coupled to U.

In devices such as provided herein, e.g., such as described withreference to any of FIG. 3A-3B, 7A-7D, 8A-8E, or 9A-9D, the firstluminophore can be coupled to a first polynucleotide to be sequenced,and the second luminophore can be coupled to a second polynucleotide tobe sequenced. For example, the first polynucleotide can be coupled tothe first feature, and the second polynucleotide can be coupled to thesecond feature. The device can further include a first polymerase addinga first nucleic acid to a third polynucleotide that is complementary toand coupled to the first polynucleotide, the first nucleic acid beingcoupled to the first luminophore. The device further can include asecond polymerase adding a second nucleic acid to a fourthpolynucleotide that is complementary to and coupled to the secondpolynucleotide, the second nucleic acid being coupled to the secondluminophore. The device further can include a channel flowing a firstliquid including the first and second nucleic acids and the first andsecond polymerases into or over the first and second features. Forexample, the first and second polynucleotides can be coupled to thefirst and second features that are disposed over a first pixel, and thatare to be sequenced using a suitable SBS scheme. The first and secondluminophores respectively can be coupled to first and second nucleicacids that respectively are being incorporated into the first and secondpolynucleotides, e.g., using the first and second polymerases. Followingan SBS step of incorporating the first and second nucleic acids into thefirst and second polynucleotides, the first and second luminophoresselectively can be luminescently imaged at different times than oneanother in a manner such as provided herein, so as to obtain respectiveelectrical signals responsive to presence of the first luminophore atthe first polynucleotide (that is, incorporation of the first nucleicacid into the first polynucleotide) and responsive to presence of thesecond luminophore at the second polynucleotide (that is, incorporationof the second nucleic acid into the second polynucleotide).

It should be appreciated that any suitable method can be used so as toimage luminophores at multiple sites using a given pixel. For example,FIG. 10 illustrates an exemplary flow of steps in a method providedherein for use in luminescent imaging. Method 1000 illustrated in FIG.10 can include providing an array of imaging pixels (1001). For example,arrays of imaging pixels are commercially available. Method 1000illustrated in FIG. 10 also can include providing a photonic structuredisposed over the array of imaging pixels (1002). For example, aphotonic crystal, a photonic superlattice, a microcavity array, or anarray of plasmonic nanoantennas can be disposed over the array ofimaging pixels using any suitable combination of materials fabricationand patterning techniques such as known in the art.

Method 1000 illustrated in FIG. 10 also can include providing an arrayof features disposed over the photonic structure (1003). For example, anarray of wells or posts can be disposed over the photonic structureusing any suitable combination of materials fabrication and patterningtechniques such as known in the art. The array of features can beregistered with the photonic crystal and with the array of pixels suchthat an integer number n>2 of features is disposed over each pixel. Forexample, a first feature of the array of features can be disposed over afirst pixel of the array of imaging pixels, and a second feature of thearray of features can be disposed over the first pixel and spatiallydisplaced from the first feature. For example, the second feature can belaterally displaced from the first feature. In one non-limiting example,the photonic structure includes a hexagonal lattice, and the imagingpixels are rectangular. The first and second features, e.g., the firstand second posts or wells, optionally each can have a substantiallycircular cross-section. Optionally, the array of imaging pixels, thephotonic structure, and the array of features can be monolithicallyintegrated with one another, e.g., can be prepared as a unitarystructure, such as using a sequence of CMOS processing steps.

Method 1000 illustrated in FIG. 10 also can include providing a firstluminophore disposed within or over the first feature (1004), andproviding a second luminophore disposed within or over the secondfeature (1005). For example, the array of features can include aplurality of wells. The first feature can include a first well withinwhich the first luminophore is disposed, and the second feature caninclude a second well within which the second luminophore is disposed.As another example, the array of features can include a plurality ofposts. The first feature can include a first post upon which the firstluminophore is disposed, and the second feature can include a secondpost upon which the second luminophore is disposed. Optionally, thefirst and second luminophores respectively can be coupled directly orindirectly to the first and second features. As one nonlimiting example,the first and second luminophores respectively can be coupled to firstand second nucleic acids and/or can be coupled to first and secondpolynucleotides being sequenced in a manner such as described elsewhereherein.

Method 1000 illustrated in FIG. 10 also can include generating by aradiation source first photons having a first characteristic at a firsttime (1006). The first photons having the first characteristic cangenerate a first resonant pattern within the photonic structure at thefirst time, the first resonant pattern selectively exciting the firstluminophore relative to the second luminophore. Method 1000 illustratedin FIG. 10 generating by the radiation source second photons having asecond characteristic at a second time (1007). The second characteristiccan be different than the first characteristic, and the second time canbe different than the first time. In one example, steps 1006 and 1007respectively can include flood illuminating the photonic structure withthe first and second photons and/or can include generating the first andsecond photons with a laser. Optionally, the first and second photonscan be in the visible range of the spectrum and/or independently canhave wavelengths between about 300 nm and about 800 nm.

The second photons having the second characteristic can generate asecond resonant pattern within the photonic structure at the secondtime, the second resonant pattern selectively exciting the secondluminophore relative to the first luminophore. Exemplary radiationsources, photon characteristics, and resonant patterns are describedelsewhere herein. Illustratively, the first and second photoncharacteristics can be selected independently from the group consistingof wavelength, polarization, and angle. As one example, the first photoncharacteristic can include a first linear polarization, and the secondphoton characteristic can include a second linear polarization that isdifferent than the first linear polarization. Optionally, the firstlinear polarization is substantially orthogonal to the second linearpolarization. Alternatively, the first linear polarization can berotated relative to the second linear polarization by between about 15degrees and about 75 degrees. As another example, the first photoncharacteristic can include a first wavelength, and the second photoncharacteristic can include a second wavelength that is different thanthe first wavelength.

Optionally, the first and second photons each irradiate the photonicstructure at substantially the same angle as one another. For example,the first and second photons each can irradiate the photonic structureat an angle approximately normal to a major surface of the photonicstructure. Or, for example, the first and second photons each irradiatethe photonic structure at an angle approximately parallel to a majorsurface of the photonic structure. In some embodiments, the radiationsource can include an optical component, and method 1000 can furtherinclude controlling the optical component so as to impose the firstcharacteristic on the first photons and configured to impose the secondcharacteristic on the second photons. Illustratively, the opticalcomponent can include a birefringent material rotating the first photonsto a first linear polarization responsive to a first control signal by acontroller, and rotating the second photons to a second linearpolarization responsive to a second control signal by the controller.Additionally, or alternatively, the optical component can control awavelength or angle of the first and second photons responsive tocontrol signals by the controller.

Method 1000 illustrated in FIG. 10 also can include selectivelyreceiving by the first pixel luminescence emitted by the firstluminophore responsive to the first photons at the first time (1008) andselectively receiving by the first pixel luminescence emitted by thesecond luminophore responsive to the second photons at the second time(1009). In a manner such as described elsewhere herein, the first pixelcan generate respective electrical signals at the first and second timesbased upon which the first and second luminophores can be distinguishedfrom one another.

Any suitable number of features can be disposed over the first pixel,e.g., three or four features in a manner such as described herein withreference to FIG. 3A-3B, 7A-7D, 8A8E, or 9A-9D. For example, a thirdfeature of the array of features optionally can be disposed over thefirst pixel and spatially displaced from each of the first and secondfeatures, and method 1000 further can include providing a thirdluminophore disposed within or over the third feature; generating thirdphotons having a third characteristic at a third time, the thirdcharacteristic being different than the first and secondcharacteristics, the third time being different than the first andsecond times; and selectively receiving by the first pixel luminescenceemitted by the third luminophore responsive to the third photons at thethird time. Optionally, a fourth feature of the array of features can bedisposed over the first pixel and spatially displaced from each of thefirst, second, and third features, and method 1000 further can includeproviding a fourth luminophore disposed within or over the fourthfeature; generating fourth photons having a fourth characteristic at afourth time, the fourth characteristic being different than the first,second, and third characteristics, the fourth time being different thanthe first, second, and third times; and selectively receiving by thefirst pixel luminescence emitted by the fourth luminophore responsive tothe fourth photons at the fourth time. In one non-limiting example, thefirst luminophore can be coupled to a first nucleic acid, the secondluminophore can be coupled to a second nucleic acid, the thirdluminophore can be coupled to a third nucleic acid, and the fourthluminophore can be coupled to a fourth nucleic acid.

Additionally, or alternatively, any suitable number of features can bedisposed over a second pixel, e.g., two, three, four, or more than fourfeatures. For example, a third feature of the array of features can bedisposed over a second pixel of the array of imaging pixels; a fourthfeature of the array of features can be disposed over the second pixeland spatially displaced from the third feature; and method 1000 furthercan include providing a third luminophore disposed within or over thethird feature; and providing a fourth luminophore disposed within orover the fourth feature. Method 1000 further can include selectivelyreceiving by the second pixel luminescence emitted by the thirdluminophore responsive to the first photons at the first time orresponsive to the second photons at the second time; and selectivelyreceiving by the second pixel luminescence emitted by the fourthluminophore responsive to the first photons at the first time orresponsive to the second photons at the second time. In one non-limitingexample, the first luminophore can be coupled to a first nucleic acid,the second luminophore can be coupled to a second nucleic acid, thethird luminophore can be coupled to a third nucleic acid, and the fourthluminophore can be coupled to a fourth nucleic acid.

Method 1000 can be adapted for luminescent imaging in an SBS scheme. Forexample, method 1000 can include providing at least one microfluidicfeature in contact with the array of features and flowing, by the atleast one microfluidic feature, one or more analytes to the first andsecond features. As another example, the first luminophore can becoupled to a first nucleotide, and the second luminophore can be coupledto a second nucleotide. Additionally, or alternatively, the firstluminophore is coupled to a first polynucleotide to be sequenced, andthe second luminophore can be coupled to a second polynucleotide to besequenced. The first polynucleotide can be coupled to the first feature,and the second polynucleotide can be coupled to the second feature.Method 1000 further can include adding, by a first polymerase, a firstnucleotide to a third polynucleotide that is complementary to andcoupled to the first polynucleotide, the first nucleotide being coupledto the first luminophore. Method 1000 further can include adding, by asecond polymerase, a second nucleotide to a fourth polynucleotide thatis complementary to and coupled to the second polynucleotide, the secondnucleotide being coupled to the second luminophore. Method 1000 furthercan include flowing, by a channel, a first liquid including the firstand second nucleotides and the first and second polymerases into or overthe first and second features.

As noted elsewhere herein, the present devices and methods can beprepared using any suitable combination of materials processing andpatterning techniques. FIG. 11 illustrates an exemplary sequence ofsteps that can be used to prepare a device or composition such asprovided herein. Illustratively, a device composition such as describedherein with reference to FIGS. 3A-3B can be prepared using twonanoimprint lithography steps, followed by conformal dielectricdeposition steps. For example, at step (a) of FIG. 11 , a first,optically transparent material such as a dielectric or a semiconductor,e.g., a polymer (such as a resin), can be disposed over a substrate,e.g., a glass substrate. At step (b) of FIG. 11 , the first material canbe patterned using nanoimprint lithography, e.g., so as to define aplurality of features, such as wells or posts. At step (c) of FIG. 11 ,a third, optically transparent material, e.g., a dielectric orsemiconductor material having a higher refractive index than the firstmaterial, can be disposed (e.g., conformally coated) over the featuresdefined within the first material. At step (d) of FIG. 11 , a fourthoptically transparent material such as a dielectric or semiconductor,e.g., polymer (such as a resin) can be disposed over the third material.At step (e) of FIG. 11 , the fourth material can be patterned usingnanoimprint lithography, e.g., so as to define a plurality of wells ornanowells. The fourth material optionally fills spaces within thephotonic structure, such as illustrated in FIG. 11(e). A second material(not specifically illustrated) that includes one or more luminophorescan be disposed within the wells or nanowells, e.g., can be disposedover the photonic structure. Although FIG. 11 illustrates preparation ofa single well per pixel, it should be understood that multiple wells perpixel readily can be prepared, e.g., by changing the distribution andsize of features formed in the second nanoimprint step.

As another example, a device or composition such as described hereinwith reference to FIGS. 3A-3B can be prepared using a combination of twophotolithography steps, two RIE steps, a dielectric deposition and CMP.For example, FIG. 12 illustrates another exemplary sequence of stepsthat can be used to prepare a device or composition such as providedherein. At step (a) of FIG. 12 , a first, optically transparent materialsuch as a dielectric or a semiconductor, e.g., a polymer (such as aresin), can be disposed over a substrate, e.g., a glass substrate. Atstep (b) of FIG. 12 , the first material can be patterned usingphotolithography followed by reactive ion etch (RIE), e.g., so as todefine a plurality of features, such as wells or posts. At step (c) ofFIG. 12 , a third, optically transparent material, e.g., a dielectric orsemiconductor material having a higher refractive index than the firstmaterial, can be disposed (e.g., conformally coated) over the featuresdefined within the first material. At step (d) of FIG. 12 , a fourthoptically transparent material such as a dielectric or semiconductor,e.g., polymer (such as a resin) can be disposed over the third material.At step (e) of FIG. 12 , the fourth material can be planarized, e.g.,using chemical mechanical polishing (CMP). At step (f) of FIG. 12 , thefourth material can be patterned using photolithography followed by RIE,e.g., so as to define a plurality of wells or nanowells. The fourthmaterial optionally fills spaces within the photonic structure, such asillustrated in FIG. 12(f). A second material (not specificallyillustrated) that includes one or more luminophores can be disposedwithin the wells or nanowells, e.g., can be disposed over the photonicstructure. Although FIG. 12 illustrates preparation of a single well perpixel, it should be understood that multiple wells per pixel readily canbe prepared, e.g., by changing the distribution and size of featuresformed in the second lithography and RIE steps.

It should be understood that the present devices suitably can be used inany of a variety of applications, e.g., for luminescent imaging. Forexample, FIG. 13 illustrates an exemplary device for use in luminescentimaging such as provided herein. FIG. 13 illustrates an exemplary devicethat includes photonic structure 1310, optical component 1330, imagingpixel 1350, and detection circuit 1340. Photonic structure 1310 includesa first material (indicated by diagonal pattern) having a firstrefractive index, and a second material (indicated by horizontally linedpattern) having a second refractive index that is different than thefirst refractive index. The first material can include first and secondmajor surfaces 1311, 1312, and first and second pluralities of features,e.g., wells, 1313, 1314 defined through at least one of the first andsecond major surfaces. The features, e.g., wells, of the first plurality1313 optionally can differ in at least one characteristic from thefeatures, e.g., wells, of the second plurality 1314, e.g., can differ inshape, size, or distribution. For example, in the exemplary photonicstructure 1310 illustrated in FIG. 13 , the features, e.g., wells, ofthe first plurality 1313 optionally can differ in size (e.g., width) andin distribution (e.g., spacing) as compared to the features of thesecond plurality 1314. In the nonlimiting example illustrated in FIG. 13, the second material can be disposed within or between the first andsecond pluralities of features, e.g., wells, 1313, 1314 and can includefirst and second luminophores 1321, 1322. For example, some of first andsecond luminophores 1321, 1322 can be located within or between thefirst plurality of features, e.g., wells, 1313, and other of the firstand second luminophores 1321, 1322 can be located within or between thesecond plurality of features, e.g., wells, 1314. In other embodimentssuch as discussed above with reference to FIGS. 3A-3B, the secondmaterial can be disposed over the first and second pluralities offeatures. Illustratively, the first material can include a polymer or aglass or other suitable material, or the second material can include afluid or a gel or other suitable material. Optionally, photonicstructure 1310 further includes a third material having a thirdrefractive index that is different than the first and second refractiveindices, the third material being disposed over at least one of thefirst and second pluralities of features, the second material beingdisposed over the third material, in a manner such as described hereinwith reference to FIGS. 3A-3B. Optionally, first luminophore 1321 can becoupled to a first nucleic acid, and second luminophore 1322 can becoupled to a second nucleic acid that is different than the firstnucleic acid.

Photonic structure 1310 can selectively support a first resonant patternresponsive to irradiation with photons having a first characteristic ata first time, responsive to which first luminophore 1321 can emit firstwavelength λ₁. Photonic structure 1310 can selectively support a secondresonant pattern responsive to irradiation with photons having a secondcharacteristic at a second time, responsive to which second luminophore1322 can emit and second wavelength λ₂. The first and second wavelengthsoptionally can be different from one another, e.g., optionally can beseparated from one another by a first non-propagating wavelength thatdoes not selectively resonate within the photonic structure. Opticalcomponent 1330 can be disposed over one of the first and second majorsurfaces 1311, 1312 of the first material, e.g., over and optionally ata spaced distance from first major surface 1311. Optical component 1330can be configured so as to irradiate photonic structure 1310 with thefirst photons at the first time and to irradiate photonic structure 1310with the second photons at the second time. In the exemplary deviceillustrated in FIG. 13 , the photonic structure 1310 is irradiated withthe first and second photons at an angle approximately normal to firstmajor surface 1311 of the first material, but it should be understoodthat any other angle, such as an angle disclosed herein, suitably can beused.

Photonic structure 1310 can be disposed over imaging pixel 1350, whichcan include an image sensor configured to image the received first andsecond wavelengths λ₁, λ₂ at the first and second times, respectively.Pixel 1350 can be spaced apart from photonic structure 1310, or can bein contact with photonic structure 1310, e.g., can be disposed incontact with second major surface 1312. Illustratively, pixel 1350 caninclude a complementary metal-oxide semiconductor (CMOS) based imagesensor in contact with photonic structure 1310. Detection circuit 1340,which can be suitably electronically coupled to pixel 1350, can beconfigured so as to receive and analyze electrical signals from pixel1350 at the first and second times. In a nonlimiting example in whichthe first and second luminophores respectively are coupled to first andsecond nucleic acids, detection circuit 1340 can be configured so as toidentify, based on the electrical signals at the first and second times,which of the first and second nucleic acids have been to a particularpolynucleotide that is coupled to the photonic structure, e.g., in amanner such as described elsewhere herein. Other imaging pixels, such asa pixel of a CCD camera, can be used. Exemplary detectors are set forthin Bentley et al., Nature 456:53-59 (2008), PCT Publ. Nos. WO 91/06678,WO 04/018497 or WO 07/123744; U.S. Pat. Nos. 7,057,026, 7,329,492,7,211,414, 7,315,019 or 7,405,281, and US Pat. App. Publ. No.2008/0108082, the entire contents of each of which is incorporatedherein by reference.

Devices such as provided herein further can transmit radiation to thephotonic structure so as suitably to excite luminophores therein. Forexample, device 1300 further can include a broadband excitation source,such as a light emitting diode (LED), or a narrowband excitation source,such as a laser, configured to generate radiation transmitted to thephotonic structure by optical component 1330.

Note that the present devices, such as device 1300 illustrated in FIG.13 , optionally can include one or more microfluidic features such asdescribed elsewhere herein. For example, device 1300 optionally caninclude at least one microfluidic feature in contact with the photonicstructure and configured to provide a flow of one or more analytes into,between, or over the first and second pluralities of features disposedover the pixel. Such analytes optionally can include one or morereagents for nucleic acid sequencing such as nucleotides, nucleic acidsor polymerases.

Thus, provided herein are devices, compositions, and methods includingphotonic structures that can provide single color or multicolorluminescence signal enhancement at a greater number of sites than thenumber of pixels used in luminescent imaging, e.g., are compatible withpreviously known epifluorescence microscopy scanning systems, such assequencing platforms that are commercially available, e.g., fromIllumina, Inc. For example, some embodiments of the present devices,compositions, and methods can create excitation “hotspots” separated bydistances on the order of the wavelength of light. Spatial distributionof these high-intensity resonant features (e.g., Fano or guided moderesonances) can be tuned, for example, by appropriately selecting thephotonic structure lattice features (e.g., symmetry) and/or thewavelength, angle, and or/polarization state of the excitation beam.Placing luminophores (e.g., biomolecules coupled to such luminophores)in proximity to such photonic structures can enhance luminescence signalbut resonantly enhancing luminophore excitation, luminescencecollection, or both. As such, photonic structures are an attractiveplatform for achieving luminescence signal enhancement from multipleimaging sites above single pixels, e.g., using uniform illumination,where selective imaging site excitation can be achieved by controllingthe characteristics of the excitation beam at different times. Thephotonic structures can be tuned so as to reduce cross-talk terms suchas described herein with reference to FIGS. 7D and 8E. Alternatively,the photonic structure can be omitted, and the excitation beam can bedirected to selected ones of the imaging sites, e.g., using free spaceoptics or multi-laser interference.

Other Alternative Embodiments

While various illustrative embodiments of the invention are describedabove, it will be apparent to one skilled in the art that variouschanges and modifications may be made therein without departing from theinvention. For example, although certain compositions, systems, andmethods are discussed above with reference to luminescent imagingassociated with sequencing polynucleotides such as DNA or RNA, it shouldbe understood that the present compositions, systems, and methodssuitably can be adapted for use in luminescent imaging associated withany appropriate subject. The appended claims are intended to cover allsuch changes and modifications that fall within the true spirit andscope of the invention.

What is claimed:
 1. A device for use in luminescent imaging, the devicecomprising: an array of imaging pixels; a photonic structure disposedover the array of imaging pixels; an array of wells disposed over thephotonic structure, the array of wells comprising: a first wellcorresponding to a first pixel of the array of imaging pixels, and asecond well corresponding to the first pixel, the second well beingspatially displaced from the first well; a first luminophore disposedwithin the first well; a second luminophore disposed within the secondwell; a radiation source configured to generate first photons having afirst characteristic, and configured to generate second photons having asecond characteristic, the second characteristic being different thanthe first characteristic, wherein the first and second characteristicsare selected independently from the group consisting of polarization,wavelength, and angle; and a controller coupled to the radiation sourceand configured to cause the radiation source to illuminate the photonicstructure with the first photons at a first time and to illuminate thephotonic structure with the second photons at a second time which isdifferent from the first time, wherein the photonic structure causes thefirst photons to interfere with one another in such a manner as toselectively illuminate the first well, with a first interferencepattern, the first interference pattern having a field strength that ismore intense at the first well than at the second well and thusselectively excites the first luminophore to a greater extent than thesecond luminophore at the first time, and wherein the photonic structurecauses the second photons to interfere with one another in such a manneras to selectively illuminate the second well, with a second interferencepattern, the second interference pattern having a field strength that ismore intense at the second well than at the first well and thus excitesthe second luminophore to a greater extent than the first luminophore atthe second time, the second interference pattern being different thanthe first interference pattern; and wherein the first pixel isconfigured to receive luminescence emitted by the first luminophore ofthe first well responsive to the selective illumination of the firstwell by the first interference pattern at the first time, and isconfigured to receive luminescence emitted by the second luminophore ofthe second well responsive to selective illumination of the second wellby the second interference pattern at the second time.
 2. The device ofclaim 1, wherein the array of imaging pixels, the photonic structure,and the array of wells are monolithically integrated with one another.3. The device of claim 1, wherein the photonic structure comprises aphotonic crystal, a photonic superlattice, a microcavity array, or anarray of plasmonic nanoantennas.
 4. The device of claim 1, wherein thesecond well is laterally displaced from the first well.
 5. The device ofclaim 1, wherein: a third well of the array of wells corresponds to thefirst pixel and is spatially displaced from each of the first and secondwells; the device further comprises a third luminophore disposed withinthe third well; the radiation source is configured to generate thirdphotons having a third characteristic, the third characteristic beingdifferent than the first and second characteristics; the controller isconfigured to cause the radiation source to illuminate the photonicstructure with the third photons at a third time, the third time beingdifferent than the first and second times; the photonic structure causesthe third photons to interfere with one another in such a manner as toselectively illuminate the third well, with a third interferencepattern, the third interference pattern having a field strength that ismore intense at the third well than at the first and second wells andthus selectively excites the third luminophore to a greater extent thanthe first and second luminophores at the third time, the thirdinterference pattern being different from the first and secondinterference patterns; and the first pixel is configured to receiveluminescence emitted by the third luminophore of the third wellresponsive to the selective illumination of the third well by the thirdinterference pattern at the third time.
 6. The device of claim 5,wherein: a fourth well of the array of wells corresponds to the firstpixel and is spatially displaced from each of the first, second, andthird wells; the device further comprises a fourth luminophore disposedwithin the fourth well; the radiation source is configured to generatefourth photons having a fourth characteristic, the fourth characteristicbeing different than the first, second, and third characteristics; thecontroller is configured to cause the radiation source to illuminate thephotonic structure with the fourth photons at a fourth time, the fourthtime being different than the first, second, and third times; thephotonic structure causes the fourth photons to interfere with oneanother in such a manner as to selectively illuminate the fourth wellwith a fourth interference pattern, the fourth interference patternhaving a field strength that is more intense at the fourth well than atthe first, second, and third wells and thus selectively excites thefourth luminophore to a greater extent than the first, second, and thirdluminophores at the fourth time, the fourth interference pattern beingdifferent from the first, second, and third interference patterns; andthe first pixel is configured to receive luminescence emitted by thefourth luminophore of the fourth well responsive to the selectiveillumination of the fourth well by the fourth interference pattern atthe fourth time.
 7. The device of claim 1, wherein: a third well of thearray of wells corresponds to a second pixel of the array of imagingpixels; a fourth well of the array of wells corresponds to the secondpixel and is spatially displaced from the first, second, and thirdwells; the device further comprises a third luminophore disposed withinthe third well; the device further comprises a fourth luminophoredisposed within the fourth well; the first interference pattern furtherselectively illuminates the third well, the first interference patternhaving a field strength that is more intense at the third well than atthe fourth well and thus selectively excites the third luminophore to agreater extent than the fourth luminophore at the first time; the secondinterference pattern further selectively illuminates the fourth well,the second interference pattern having a field strength that is moreintense at the fourth well than at the third well and thus selectivelyexcites the fourth luminophore to a greater extent than the thirdluminophore at the second time; the second pixel is configured toreceive luminescence emitted by the third luminophore responsive to theselective illumination of the third well by the first interferencepattern at the first time; and the second pixel is configured to receiveluminescence emitted by the fourth luminophore responsive to theselective illumination of the fourth well by the second interferencepattern at the second time.
 8. The device of claim 1, wherein thephotonic structure comprises a hexagonal lattice and wherein the imagingpixels are rectangular.
 9. The device of claim 1, wherein the first andsecond photons independently have wavelengths between about 300 nm andabout 800 nm.
 10. The device of claim 1, wherein the first luminophoreis coupled to a first nucleic acid, wherein the second luminophore iscoupled to a second nucleic acid, wherein the first nucleic acid iscoupled to a first polynucleotide to be sequenced, and wherein thesecond nucleic acid is coupled to a second polynucleotide to besequenced.
 11. A method for use in luminescent imaging, the methodcomprising: at a first time, illuminating a photonic structure disposedover an array of imaging pixels with first photons from a radiationsource, the first photons having a first characteristic; causing, by thephotonic structure, the first photons to interfere with one another insuch a manner as to selectively illuminate a first well of an array ofwells disposed over the photonic structure, with a first interferencepattern, the first interference pattern having a field strength that ismore intense at the first well than at a second well of the array ofwells, the first well corresponding to a first pixel of the array ofimaging pixels; at a second time, illuminating the photonic structurewith second photons from the radiation source, the second photons havinga second characteristic, the second characteristic being different thanthe first characteristic, the second time being different than the firsttime, wherein the first and second characteristics are selectedindependently from the group consisting of polarization, wavelength, andangle; causing, by the photonic structure, the second photons tointerfere with one another in such a manner as to selectively illuminatethe second well, with a second interference pattern, the secondinterference pattern having a field strength that is more intense at thesecond well than at the first well, the second well corresponding to thefirst pixel and spatially displaced from the first well, the secondinterference pattern being different than the first interferencepattern; receiving, by the first pixel, luminescence emitted by a firstluminophore disposed within the first well responsive to the selectiveillumination of the first well by the first interference pattern at thefirst time; and receiving, by the first pixel, luminescence emitted by asecond luminophore disposed within the second well responsive to theselective illumination of the second well by the second interferencepattern at the second time.
 12. The method of claim 11, wherein thephotonic structure comprises a photonic crystal, a photonicsuperlattice, a microcavity array, or an array of plasmonicnanoantennas.
 13. The method of claim 11, wherein the second well islaterally displaced from the first well.
 14. The method of claim 11,wherein a third well of the array of wells corresponds to the firstpixel and is spatially displaced from each of the first and secondwells, and the method further comprises: at a third time, illuminatingthe photonic structure with third photons from the radiation source, thethird photons having a third characteristic, the third characteristicbeing different than the first and second characteristics, the thirdtime being different than the first and second times; causing, by thephotonic structure, the third photons to interfere with one another insuch a manner as to selectively illuminate the third well, with a thirdinterference pattern, the third interference having a field strengththat is more intense at the third wells than at the first and secondwells, the third well being spatially displaced from the first andsecond wells, the third interference pattern being different than thefirst and second interference patterns; and receiving, by the firstpixel, luminescence emitted by a third luminophore disposed within thethird well responsive to the selective illumination of the third well bythe third interference pattern at the third time.
 15. The method ofclaim 11, wherein: a third well of the array of wells corresponds to asecond pixel of the array of imaging pixels; a fourth well of the arrayof wells corresponds to the second pixel and is spatially displaced fromthe third well; and the method further comprises: receiving, by thesecond pixel, luminescence emitted by a third luminophore disposedwithin the third well responsive to selective illumination of the thirdwell by the first interference pattern at the first time; and receiving,by the second pixel, luminescence emitted by a fourth luminophoredisposed within the fourth well responsive to selective illumination ofthe fourth well by the second interference pattern at the second time.16. The method of claim 11, comprising flood illuminating the photonicstructure with the first photons at the first time and with the secondphotons at the second time.
 17. The method of claim 11, furthercomprising flowing one or more analytes to the first and second wells.18. The device of claim 10, further comprising: a third polynucleotide,the third polynucleotide being complementary to and coupled to the firstpolynucleotide; a fourth polynucleotide, the fourth polynucleotide beingcomplementary to and coupled to the second polynucleotide; a firstpolymerase, the first polymerase being configured to add the firstnucleic acid to the third polynucleotide, the first polynucleotide beingcoupled to the first well; a second polymerase, the second polymerasebeing configured to add the second nucleic acid to the fourthpolynucleotide, the second polynucleotide being coupled to the secondwell; a liquid, the liquid comprising the first and second nucleic acidsand the first and second polymerases; and a channel, the channel beingconfigured to allow the liquid to flow through and into or over thefirst and second wells.
 19. The method of claim 11, wherein the firstluminophore is coupled to a first nucleic acid, wherein the secondluminophore is coupled to a second nucleic acid, wherein the firstnucleic acid is coupled to a first polynucleotide to be sequenced, andwherein the second nucleic acid is coupled to a second polynucleotide tobe sequenced.
 20. The device of claim 3, wherein the photonic structurecomprises a photonic superlattice, the photonic superlattice supportingpropagation of the first photons and the second photons, the photonicsuperlattice inhibiting propagation of third photons having a wavelengthbetween the wavelength of the first photons and the wavelength of thesecond photons.
 21. The device of claim 3, wherein the photonicstructure comprises a photonic superlattice, the photonic superlatticecomprising: a first material having a first refractive index, the firstmaterial comprising first and second major surfaces and first and secondpluralities of wells defined through at least one of the first andsecond major surfaces, the wells of the first plurality differing in atleast one characteristic from the wells of the second plurality; and asecond material having a second refractive index that is different thanthe first refractive index, the second material being disposed within,between, or over the first and second pluralities of wells andcomprising the first and second luminophores.
 22. The method of claim12, wherein the photonic structure comprises a photonic superlattice,the photonic superlattice supporting propagation of the first photonsand the second photons, the photonic superlattice inhibiting propagationof third photons having a wavelength between the wavelength of the firstphotons and the wavelength of the second photons.
 23. The method ofclaim 12, wherein the photonic structure comprises a photonicsuperlattice, the photonic superlattice comprising: a first materialhaving a first refractive index, the first material comprising first andsecond major surfaces and first and second pluralities of wells definedthrough at least one of the first and second major surfaces, the wellsof the first plurality differing in at least one characteristic from thewells of the second plurality; and a second material having a secondrefractive index that is different than the first refractive index, thesecond material being disposed within, between, or over the first andsecond pluralities of wells and comprising the first and secondluminophores.
 24. The method of claim 10, wherein the photonic structurecomprises a hexagonal lattice and wherein the imaging pixels arerectangular.
 25. The method of claim 19, further comprising: adding, bya first polymerase, a first nucleic acid to a third polynucleotide thatis complementary to and coupled to the first polynucleotide, the firstnucleic acid being coupled to the first luminophore, and the firstpolynucleotide being coupled to the first well; adding, by a secondpolymerase, a second nucleic acid to a fourth polynucleotide that iscomplementary to and coupled to the second polynucleotide, the secondnucleic acid being coupled to the second luminophore, and the secondpolynucleotide being coupled to the second well; and flowing, by achannel, a liquid including the first and second nucleic acids and thefirst and second polymerases into or over the first and second wells.